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ω-Transaminase in Biocatalysis
Methods, Reactions and Engineering
Karim Engelmark Cassimjee
Doctoral thesis
KTH Royal Institute of Technology
School of Biotechnology
Stockholm 2012
i
Cover picture:
The Enzyme at the Heart of the Green Chemical Factory
© David Karim Engelmark Cassimjee 2012
KTH Royal Institute of Technology
School of Biotechnology
AlbaNova University Centre
SE-106 91 Stockholm
Sweden
ISBN 978-91-7501-242-1
TRITA-BIO Report 2012:13
ISSN 1654-2312
Printed in Stockholm, March 2012
Universitetsservice US-AB
Drottning Kristinas väg 53 B
SE-100 44 Stockholm
Sweden
ii
For Nadia, my one year old daughter, who I suspect will enjoy
tearing out the pages (especially the ones with figures).
iii
Abstract
Biocatalysis offers an alternative to classic chemistry by using
enzymes, the protein catalysts of Nature, for production of fine
chemicals. Evolution has created enzymes capable of catalysis at
moderate temperature of a specific reaction in the presence of a
plethora of compounds in the aqueous cell environment. The focal
point of biocatalysis is to utilise these traits in vitro, for creation of
valuable molecules.
The ω-transaminase is an enzyme capable of producing
chiral amines, compounds used to great extent in pharmaceuticals.
Much effort has in recent years been invested in the research and
engineering of this enzyme type since the catalysed reaction offers
an advantageous alternative to classical techniques. Nevertheless,
there is a need for method development, adaptation of the enzyme
and increased understanding of the catalytic mechanism for
feasibility as an effective biocatalyst for unnatural substrates. This
thesis addresses a chosen set of obstacles as a contribution to
meeting
the
demands
at
hand.
ω-Transaminase
from
Chromobacterium violaceum and Arthrobacter citreus was used.
Many homologous ω-transaminases are available, which are
also subject to engineering where variants are produced. To
accurately compare their kinetic constants an active site
quantification method is required but has not been available. Here
such a method is presented (Paper 1) which encompasses a virtually
irreversible half transamination reaction.
In stereoselective synthesis the ω-transaminase catalysed
equilibrium reaction inherently results in incomplete conversion. An
equilibrium displacement system is presented (Paper II) where
isopropylamine is the amino donor for transamination of
acetophenone and derivatives thereof, coupled to an enzymatic
cascade reaction.
For many unnatural substrates the specificity and
enantiospecificity is insufficient. Rationally redesigned variants were
produced with improved properties for chosen substrates (Paper III
and IV). The catalytic contributions of field and resonance of a
variant compared to the wild type were investigated (Paper IV) for
increased knowledge of the mechanism.
For rational redesign of an enzyme the three-dimensional
structure is required, of which only a few are available for the ωtransaminases. X-ray crystallographic structures of the holo and apo
form of Chromobacterium violaceum ω-transaminase were made
(Paper V) which revealed significant structural rearrangements upon
coenzyme binding which may be of consequence for future
engineering.
iv
Sammanfattning
Biokatalys utgör ett alternativ till klassisk kemi genom att
finkemikalier produceras med hjälp av enzymer, naturens
katalysatorer. Naturen har genom evolution skapat enzymer med
förmåga att vid måttlig temperatur katalysera en specifik reaktion i
närvaro av ett överflöd av föreningar i vattenmiljön hos den levande
cellen. Huvudsyftet med biokatalys är att utnyttja dessa egenskaper
in vitro, för att producera värdefulla molekyler.
ω-Transaminaset är ett enzym med förmåga att producera
kirala aminer, föreningar som används i läkemedelsutveckling i stor
omfattning. Mycket forskning har under de senaste åren investerats
i detta enzymslag eftersom den katalyserade reaktionen utgör ett
fördelaktigt alternativ till klassiska tekniker. Dock finns det behov
av metodutveckling, anpassning av enzymet och ökad förståelse för
den katalytiska mekanismen för dess effektiva användning som
biokatalysator för onaturliga substrat. Denna avhandling behandlar
valda begränsande faktorer, ett bidrag till att möta de krav som
ställs. ω-Transaminaser från Chromobacterium violaceum och
Arthrobacter citreus användes.
Många homologa ω-transaminaser finns tillgängliga, vilka
också är föremål för utveckling av enzymvarianter. För att jämföra
kinetiska data mellan dessa är en metod för kvantifiering av aktiva
säten nödvändig. En sådan metod presenteras (Artikel I) som
innefattar en synbart irreversibel halv-transamineringsreaktion.
I stereoselektiv syntes med ω-transaminas resulterar den
katalyserade jämviktsreaktionen i ofullständig omsättning. Ett
jämviktsförskjutningssystem presenteras här (Artikel II) där isopropylamin är aminodonator för transamineringen av acetofenon
och dess derivat, kopplat till en enzymatisk kaskadreaktion.
För många onaturliga substrat är specificiteten och enantiospecificiteten otillräckliga. Rationellt designade varianter producerades med förbättrade egenskaper för utvalda substrat (Artikel III
och IV). De katalytiska bidragen från fält- och resonanseffekter hos
en variant jämfört med vildtypen undersöktes (Artikel IV) med syfte
att öka kunskapen om reaktionsmekanismen.
Rationell design av ett enzym fordrar dess tredimensionella
struktur, endast ett fåtal sådana är tillgängliga för ω-transaminaser.
Röntgenkristallografiska strukturer av holo- och apo-former av
Chromobacterium violaceum ω-transaminas togs fram (Artikel V),
som visade tydliga strukturella omorganiseringar vid koenzymbindning som kan vara av betydelse för framtida utveckling.
v
This thesis is based on the following
scientific publications:
I
Active Site Quantification of an ω-Transaminase by Performing
a Half Transamination Reaction
Karim Engelmark Cassimjee1, Maria Svedendahl Humble1,
Valentina Miceli, Carla Granados Colomina, and Per Berglund
ACS Catalysis 2011, 1, 1051-1055
II
Transaminations with Isopropyl Amine: Equilibrium
Displacement with Yeast Alcohol Dehydrogenase Coupled to in
situ Cofactor Regeneration
Karim Engelmark Cassimjee, Cecilia Branneby, Vahak Abedi,
Andrew Wells and Per Berglund
Chemical Communications 2010, 46, 5569-5571
III
Key Amino Acid Residues for Reversed or Improved
Enantiospecificity of an ω-Transaminase
Maria Svedendahl Humble1, Karim Engelmark Cassimjee1,
Vahak Abedi, Hans-Jürgen Federsel and Per Berglund
ChemCatChem 2012, In press, doi: 10.1002/cctc.201100487
IV
Chromobacterium violaceum ω-Transaminase Variant
Trp60Cys Shows Increased Specificity for (S)-1Phenylethylamine and 4’-Substituted Acetophenones, and
Follows Swain-Lupton Parameterisation
Karim Engelmark Cassimjee, Maria Svedendahl Humble,
Henrik Land, Vahak Abedi and Per Berglund
Submitted manuscript
V
Crystal Structures of the Chromobacterium violaceum ωTransaminase Reveal Major Structural Rearrangements Upon
Binding of Coenzyme PLP
Maria Svedendahl Humble, Karim Engelmark Cassimjee,
Maria Håkansson, Yengo Kimbung, Björn Walse, Vahak Abedi,
Hans-Jürgen Federsel, Per Berglund and Derek T. Logan
FEBS Journal 2012, 279, 779-792
The above publications will be referred to by their assigned roman
numerals.
1
Shared first authorship
vi
Summary of the contributions of Karim E. Cassimjee
(KC) to Papers I to V
Paper I: KC supplied the idea of using a virtually irreversible half
transamination reaction for active site quantification, designed and
performed the active site quantification method, designed the
molecular cloning, and performed the majority of the writing.
Paper II: KC supplied the idea of utilizing the selectivity of yeast
alcohol dehydrogenase for equilibrium displacement, carried out the
molecular cloning, reactions and analysis, and did the majority of
the writing.
Paper III: KC designed the molecular cloning and the experimental
procedures for enantioselectivity determination, and took part in the
rational redesign and the writing.
Paper IV: KC performed the molecular cloning, reactions and
analysis, reaction parameterisation, took part in the finding of the
key point mutation, and performed the majority of the writing.
Paper V: KC designed the molecular cloning, performed stability
experiments, and took part in the writing.
Publications by KC, not included in this thesis
•
One-Step Enzyme Extraction and Immobilization for
Biocatalysis Applications
Karim Engelmark Cassimjee, Robert Kourist, Diana Lindberg,
Marianne Wittrup Larsen, Nguyen Hong Thanh, Mikael
Widersten, Uwe T Bornscheuer and Per Berglund
Biotechnology Journal 2011, 6, 463-469
•
Silica-Immobilized His6-Tagged Enzyme: Alanine Racemase in
Hydrophobic Solvent
Karim Engelmark Cassimjee, Martin Trummer, Cecilia
Branneby and Per Berglund
Biotechnology and Bioengineering 2008, 99, 712-716
•
Synthesis of Cyclic Polyamines by Enzymatic Generation of an
Amino Aldehyde in situ
Karim Engelmark Cassimjee, Sílvia Rodríguez Marín and Per
Berglund
Submitted manuscript
•
Process for the Production of Polyamines
Bernhard Hauer, Karim Engelmark Cassimjee, Per Berglund
2011 Patent publication number: WO2011051433
vii
List of Abbreviations
1-PEA
Cv-ωTA
E-value
ee
Enz
eq.
GC
HPLC
IMAC
IPA
PLP
PMP
RMSD
U
1-phenylethylamine
Chromobacterium violaceum ω-transaminase
ratio of specificity constants (kcat/KM) of enantiomers
enantiomeric excess
enzyme
equation
gas chromatography
high pressure liquid chromatography
immobilised metal affinity chromatography
isopropylamine
pyridoxal-5’-phosphate
pyridoxamine-5’-phosphate
root-mean-square deviation
enzyme activity unit, defined as (µmol/min)
Amino acid (residue) abbreviations
Ala, A
alanine
Arg, R
arginine
Asn, N
asparagine
Asp, D
aspartate
Cys, C
cysteine
Glu, E
glutamate
Gln, Q
glutamine
Gly, G
glycine
His, H
histidine
Ile, I
isoleucine
Leu, L
leucine
Lys, K
lysine
Met, M
methionine
Phe, F
phenylalanine
Pro, P
proline
Ser, S
serine
Thr, T
threonine
Trp, W
tryptophan
Tyr, Y
tyrosine
Val, V
valine
viii
”A tidy laboratory means a lazy chemist.”
Jöns Jacob Berzelius (1779-1848)
Note: If the above statement is valid in the opposite sense, i.e. a messy laboratory
means a devoted chemist; my co-workers and I are possibly the most energetic
chemists in the country.
ix
Contents
1 Introduction
1.1
1.2
1.3
1.4
1 Enzymes, proteins with catalytic ability
Enantiospecificity, an enzymatic trait
Enzyme kinetics
Biocatalysis
1.4.1 Enzyme engineering
1 4 6 8 9 1.5 ω-Transaminase, a PLP-dependent enzyme
10 2 Aims and Scope
15 3 Active Site Quantification of Cv-ωTA (Paper I)
17 4 Equilibrium Displacement of ω-Transaminase
Reactions with Isopropylamine as Amino Donor
(Paper II)
21 5 Rational Redesign of Cv-ωTA (Paper III and IV)
27 5.1 Improved enantioselectivity and reversed enantiomeric
preference (Paper III)
5.2 Increased specificity (Paper IV)
29 32 6 Structure of Cv-ωTA (Paper V)
39 7 Concluding Discussion
44 7.1 Reactions and engineering with ω-transaminase,
summarising discussion of Paper I-V
7.2 Future prospects
7.2.1 Dynamic kinetic resolution of an amine
7.2.2 Mechanistic studies
45 48 48 49 8 Acknowledgements
50 9 References
51 Appendix
57 x
1 Introduction
This thesis is a contribution to the field of biocatalysis1 in which
methods, reactions and engineering with ω-transaminase are
presented. Biocatalysis is an integral part of industrial or white
biotechnology.2,3
Fine chemicals, especially pharmaceuticals, are undoubtedly
important products which have become essential for our society.
Their fabrication is a continuously developing area where there is a
demand for new and improved synthesis routes for target
compounds. A frequent motif in pharmaceuticals is the chiral
amine,4 a biofunctional group also present in natural metabolism.
In this section enzymology (with related concepts in chemistry), the
field of biocatalysis and the ω-transaminase enzyme are briefly
described. The concepts and observed traits of enzymes are put into
the context of how they may be applied for engineering and
synthesis of fine chemicals, chiral amines in particular.
1.1 Enzymes, proteins with catalytic ability
In Nature, living organisms depend on a functioning metabolism.
Cellular structures are created from simpler building blocks.
Compounds are formed and broken down in what is described as
controlled metabolic pathways in which energy is rearranged and
utilised for vital processes. The lion’s share of the chemical reactions
in these processes takes place in an aqueous environment and are
catalysed by enzymes. The name, enzyme, originates from ancient
Greek, meaning “in sourdough”. Enzymes are the foundation of
chemical life; they uphold essentially all chemical reactions at a
viable rate within a living organism.1,5
Proteins are polymers (polyamides) formed by the condensation of αamino acids (molecules which contain an amino group and a
carboxylic acid group with one methylene) which are found in all
known forms of life. Generally, residues of twenty types of amino
acid are present in proteins. The amino acids are joined in a defined
1
Figure 1: Graphical representation of an α‐helix (left) and β‐sheets (right), common motifs in proteins
order based on a gene sequence, in which each amino acid residue
contributes with a distinct side chain with properties elegantly
chosen by evolution. The proteins fold to form ordered vital
structures, biologically functional units. The structures formed by
the amino acid residues are many; the most common motifs are the
α-helix and the β-sheet which are usually depicted in threedimensional structures, shown in figure 1. When folded, the protein
molecule can form a globular structure with a hydrophobic core, a
result of minimization of hydrophobic exposure in an aqueous
environment. The individual protein molecules may exist alone or
form complexes with others. A protein which is capable of catalysing
a chemical reaction is called an enzyme.
Observed at macro scale proteins behave in unexpected ways. E.g.
with rising temperature a protein solution (such as egg white) will
turn into a semi solid (an omelette6) whereas many other compounds or solutions thereof typically melt or lower in viscosity. This
is a result of unfolding and subsequent aggregation of the protein
chains. Looking at proteins in micro scale also yields fascinating
findings. An enzyme is able to bind one or more distinct molecules
or atoms, its substrates, and provide an environment which is more
favourable for a chemical reaction of the substrate(s) than the
surrounding solution. The location in the enzyme where this takes
place is called the active site. According to the transition state
theory,7 applicable for all types of catalysis, the reaction rate
increase can be attributed to a lowering of the activation energy.
2
The mechanisms vary between enzymes and remain a matter of
debate. The theory of near attack conformers (NAC)8 presented by
Bruice and co-workers attributes the lowering of the activation
energy to the ability of an enzyme to place its substrate(s) in
positions which are favourable for reaction. Another theory,
presented by Warshel and co-workers, assigns the catalytic ability to
the presence of favourable electrostatic interactions9 in the active
site which stabilise the transition state. The enzyme macromolecule
is a flexible structure, all known enzymes display this feature.10 The
ability of conformational adaptation to favourable conformations
during a catalytic process has been presented by Hammes-Schiffer
and co-workers as being of paramount importance.10 In this view the
substrate binding, transition state stabilisation, product release,
and all transitions in-between, require different conformations of the
enzyme for effective catalysis.
Some enzymatic reactions require a non-protein moiety, a specific
molecule bound in the active site, for the catalysis to take place.
Such moieties are called coenzymes.11 When the coenzyme is bound
the complex is denoted as the holo enzyme, whereas the noncatalytic unit without coenzyme is called the apo enzyme.
Coenzymes are distinguishable from substrates since they are
regenerated, not consumed in the reaction. If the holo enzyme is
stable and the coenzyme is not degraded no more than one
equivalent of coenzyme to apo enzyme is needed for complete
activation, i.e. complete formation of holo enzyme, in a given
reaction. When non-natural conditions or non-natural substrates
are applied, arguably, there is a possibility of increased instability of
the enzyme-coenzyme complex; this should be taken into
consideration when enzymes are used in chemical synthesis. When
an enzyme catalyses an oxidation reaction a cosubstrate is used for
a complementary reduction reaction, e.g. NAD+ can be reduced to
NADH. The reversed reduction reaction will, naturally, yield NAD+
from NADH as a complementary oxidation.5 For in vitro synthesis
applications the cosubstrate may be regenerated by an auxiliary
reaction to circumvent the need for higher concentrations.
Inhibition of enzyme catalysis is a common observation, the reaction
rate may drop in the presence of a substance (an inhibitor) or higher
substrate or product concentrations.12,13 Inhibiting and activating
3
compounds are used by a living cell to control the flow of
metabolism.5 When substrate concentrations are significantly higher
than what is observed in the cell environment, arguably, inhibition
arises as a consequence of the non-natural situation. The active site
is in essence blocked by the presence of more than one substrate. In
ordered multi substrate reactions high concentrations may lead to
blockage by substrates binding in incorrect order. For in vitro
applications catalysis at high concentrations may be desired, but in
nature no evolutionary pressure existed to achieve this ability.
1.2 Enantiospecificity, an enzymatic trait
A molecule is defined as chiral if it cannot be superimposed upon its
mirror image, much alike a human hand. A molecule is not chiral
(achiral) if it has symmetry elements of the second kind, i.e. a mirror
plane, a centre of inversion, or a rotation-reflection axis. Chiral
versions of otherwise identical compounds are termed enantiomers.
In nature such molecules are predominantly existent as one of the
possible versions. In chemistry, the common way of denoting the
enantiomers are with an (S) (left handed) or (R) (right handed).
Amino acids are usually classified under a different nomenclature
which allows the majority of the in nature existing ones to be
designated as L-enantiomers, as opposed to the corresponding Denantiomers.14 An equal mix of enantiomers is referred to as a
racemate.
An enzyme is composed only of L-amino acid residues and is thereby
chiral and enantiopure. The interaction of an enzyme with chiral
substrates can differ with the enantiomers. It may possess a high
catalytic effect for one enantiomer of the substrate while the
turnover of the other is low, sometimes virtually undetectable. The
interaction of enantiomers with an enzyme can be envisioned as
analogous to hands in gloves, depicted in Figure 2.
The enantiomers may be viewed upon as different substrates.
Specificity is the term used to describe the ability of an enzyme to
distinguish between substrates, enantiospecificity when the
substrates are enantiomers. The term substrate specificity is
generally used to describe different catalytic activity for similar
substrates (not enantiomers). Enzymes also exhibit chemospecificity
4
NH2
H2N
H
H
Figure 2: A pair of human hands (the author’s) next to a right hand glove compared to the (S)‐ and (R)‐forms of 1‐phenylethylamine next to a ribbon representation of an ω‐transaminase enzyme. However rotated, either hand or molecule cannot be superimposed on the other. The right hand fits in the glove, analogous to how one of the enantiomers fits in the active site of the enzyme. for similar reactions, and regiospecificity for analogous reactions on
different regions of a substrate. Figure 3 shows examples of
substrates subject to specificity. An examination of the reactions
and compounds of metabolic pathways reveals the necessity of these
traits in order to uphold the order of metabolism. In essence, the
enzyme is evolved to catalyse a reaction specifically enough for the
remaining molecules in its environment to be sufficiently
unperturbed for continued metabolism to function sufficiently. In
this way a cell can produce one type of sugar, protein or lipid while
consuming another.
NH2
NH2
regiospecificity alcohol dehydrogenase substrate specificity ω‐transaminase OH
OH
O
N
H
chemospecificity O
amidase or esterase O
Figure 3: Examples of substrates which are subject to chemo‐, regio‐ or substrate specificity with examples of enzyme types.
5
1.3 Enzyme kinetics
When steady-state conditions can be assumed, and the enzyme
concentration is significantly lower than that of the substrate (the
usual case) the reaction rate of an enzyme catalyzed reaction can be
described by the Michaelis-Menten equation.15-19 The equation in its
simplest form, for a reaction with one substrate without inhibition,
gives:
E
M
A
A
(eq. 1)
where v is the observed reaction rate, kcat the turnover number, KM
the Michaelis-Menten constant, Enz the enzyme, and A the
substrate. From the equation follows that when the substrate
concentration is significantly lower than KM the rate is given by:
M
Enz A
(eq. 2)
by ignoring [A] in the denominator. This is the usual situation in a
living cell. When the substrate concentration is significantly higher
than KM which can then be ignored, the rate can be described by:
Enz
(eq. 3)
This is a consequence of saturation of the active sites; hence the
maximum rate denoted as Vmax. The KM is a combination of rate
constants for substrate binding, substrate dissociation and product
formation. Since the rate of product formation is generally slower
than substrate binding and dissociation, the KM can be viewed upon
as an inverse measure of the affinity of a substrate; a higher value
signifies a lower affinity. The division of the turnover number with
the Michaelis-Menten constant, kcat/KM, is a measure of the catalytic
efficiency, termed the specificity constant.12 In many cases the
specificity constant is the chosen number for comparison, rather
than the turnover number or the Michaelis-Menten constant. The
specificity constant may well have been deemed the main enzymatic
constant in the original works by Henri,15 and Michaelis and
Menten,16 where it was in fact circuitously reported as a central
factor.17 If it were, the common nomenclature would probably have
6
designated
constant.
the
specificity
constant
as
the
Michaelis-Menten
For reactions with competing substrates, multi-substrate reactions,
reactions with inhibition, etc, the rate equations become more
complex. The enzyme described in section 1.5, an ω-transaminase,
catalyses a two-substrate reaction with an ordered ping-pong bi-bi
mechanism.20 This means that two substrates enter the active site
in a defined order and the first product leaves before the second
substrate enters. The rate equation for such a situation is written
as:
E
A
M B
A B
B
M A
A B
(eq. 4)
where B is the second substrate, remaining letters follow the
definitions above. To graphically describe equation 4 a threedimensional plot is needed; experimentally many rates need to be
recorded where the two substrates are individually varied. This is
not always of practical importance; instead a pseudo one-substrate
model may be employed. Equation 4 may be rewritten as follows:
E
A
M
A
A
B
M
B
(eq. 5)
By keeping the concentration of one of the substrates at a constant
level, e.g. B in equation 5, apparent kinetic constants can be
experimentally deduced which are then valid at this chosen
concentration. The bracketed expression in the denominator of
equation 5 will be treated as a constant. This simplification is
valuable when comparisons of different substrates are made, with
the second substrate in common. When comparing enzymes or
enzyme variants pseudo one-substrate kinetics may also be applied
but the results cannot directly be regarded as general since the
affinity of the constant substrate (B in equation 5) may vary and give
arbitrary results only valid at the given reaction conditions.
However, equation 5 will simplify to equation 2 if [A] is small
enough. This means that the true specificity constant of one
substrate of an ordered ping-pong bi-bi reaction may be deduced by
studying the linear concentration range of the rate dependence with
the other substrate at a fixed concentration.
7
In a situation where two substrates are allowed to compete for an
enzyme, in a one-substrate reaction, the rate equation for an
individual substrate is given by:
A
A
E
B
M A
A
M B
B
M A
A B
M M
(eq. 6)
The ratio of the reaction rates, given by the division of equation 6 for
e.g. substrates A and B, will simplify to the division of the specificity
constants if the substrate concentrations are equal:
A
A
M
B
(eq. 7)
B
M
When the substrate is a racemate the enantiomers can be regarded
as competing substrates. The level of enantiospecificity, denoted as
the E-value, is defined as the reaction rate of the faster reacting
enantiomer divided by the rate of the slower reacting, hence the
ratio of their specificity constants as in equation 7:21,22
⁄
⁄
.
(eq. 8)
The E-value can be used for assessing the usefulness of an enzyme
for synthesizing an enantiopure compound. Henceforth, the word
enantiospecificity will be used to describe the E-value. The word
enantiopreference (enantiomeric preference) will be used to describe
which enantiomer is favoured, without regard for the degree of
specificity.
1.4 Biocatalysis
The field of biocatalysis is a merge of biology, chemistry, enzymology
and biotechnology, with an aim of using enzymes for chemical
synthesis. Theoretically, the enzymatic traits described in section
1.2 can be used for production of fine chemicals; beneficially when
other methods fail or are inefficient. Enzymes may provide the
chemist with a means of creating enantiopure compounds,
performing specific reactions in the presence of other reactive
compounds and/or steering of reactions to specific groups, in an
environmentally compatible setting.23 An enantiospecific enzyme
8
may e.g. be used for the production of an enantiopure
pharmaceutically active compound; the corresponding enantiomer
may be toxic or have an undesired effect in the human body.5,24
Classic synthetic chemistry often employs organic solvents (i.e. nonaqueous environments), high temperatures and metal catalysts.
Numerous efficient processes have been developed in this field, and
it is undoubtedly the foundation of today’s chemical industry. Still,
nature has been performing advanced chemical transformations in
aqueous environments at moderate temperature by employment of
enzymes since the beginning of life. The harnessing, mimicking and
modification of these natural processes remain an engineering
challenge, but with great promise of reward by an environmentally
sustainable chemical industry.25-27
1.4.1 Enzyme engineering
The modification of enzymes for adaptation to the environment in a
chemical process is often considered necessary, for which many
strategies have been developed.28 Often the compounds in the
process are poor substrates, the enantiospecificity may be
insufficient, or a more heat stable or co-solvent tolerable enzyme is
desired. By using an experimentally determined structure of the
enzyme (determined by X-ray chrystallography or NMR) the enzyme
engineer can locate specific amino acid residues suitable for
mutation. This is called rational redesign; arguably, the amount of
knowledge which is available regarding the function of a given
enzyme dictates the likelihood of success with this strategy. When
such attempts are unsuccessful, or a structure is unavailable, the
amino acid chain can be altered in a randomised fashion and a
library of mutants can be screened for the desired properties. An
improved clone can be further randomised and a new library can be
screened. The iterative process is repeated for the potential discovery
of the required enzyme. This strategy is called directed evolution.29-33
Further, to increase the probability of finding improved variants,
semi-rational28 approaches have been devised, such as CASTing34,35
and saturation mutagenesis. The more recent scientific publications
involving enzyme engineering contain examples of rational and semirational approaches with a higher frequency than previous years.
The method advancements, increased understanding of function
and examples of successful enzyme variants have made researchers
9
less inclined to resorting to directed evolution, which most often
involves a tedious screening procedure. The choice of method is
usually not straightforward, and remains a challenge for the enzyme
engineer.36
Notwithstanding the effectiveness of specificity (section 1.2),
enzymes may also display promiscuity,37-40 the ability to function in
a different way than for which the enzyme appear to be evolved.
Promiscuity is theorised to be a foundation for protein evolvability,41
a starting point for the emergence of beneficial properties.
When displaying condition promiscuity an enzyme may be employed
in e.g. non-aqueous environment. Substrate promiscuity can be
applied when reactions of non-natural substrates are desired.
Catalytic promiscuity, the ability to catalyse several types of similar
reactions, can be used for new desired reactions.37,38
Ample promiscuous effects arise predominantly when an enzyme is
introduced to an environment which is different from that in which
it was evolved; enzyme promiscuity is obviously not significantly
detrimental to a living cell in its natural environment. The enzyme
engineer may utilize and reinforce a desired promiscuous effect by
simply changing the solvent, or creating enzyme variants with the
methods described above.37-40,42
1.5 ω-Transaminase, a PLP-dependent enzyme
The coenzyme pyridoxal-5’-phosphate (PLP, Figure 4), a derivative of
vitamin B6, is utilized by a broad range of enzymes for catalysis of
e.g. decarboxylation, racemisation, retro-aldol cleavage and
transamination.43-45 PLP readily forms Schiff bases,14 condensation
products of amines and ketones. This feature is utilized in many
enzymes with amino substrates.43,44,46 The Schiff base structure
enables resonance stabilisation of the formed carbanion after
abstraction of, most commonly, a proton from the carbon bound to
the Schiff base nitrogen.43,44 The aldehyde in Figure 4 is the free
form of PLP, when bound to an enzyme PLP generally forms a Schiff
base with a lysine residue in the active site.44,45 The catalytic
mechanism of a given PLP-dependent enzyme can therefore be
depicted as a reaction of a Schiff base complex, the holo enzyme,
10
HO
O
O
P
O
HO
OH
N
Figure 4: The coenzyme PLP (pyridoxal‐5’‐phosphate), derivative of vitamin B6.
with the amino substrate in question; the free aldehyde in Figure 4
and its reaction with the apo enzyme is not considered a part of the
catalytic mechanism per se.
In the substrate-PLP Schiff base structure, the proton bound to the
Schiff base nitrogen is most favourably abstracted if it is positioned
anti-periplanar to the conjugated π-electron system.43,44 Therefore,
effective catalysis cannot be performed solely by the presence of
enough space for the substrate, but by the ability of the substrate to
be positioned in a catalytically productive manner. This was
demonstrated by Blackburn and co-workers by rational redesign of
an alanine racemase which was able to accept serine as substrate
when a variant enabled anti-periplanar positioning of the proton in
question.47 The situation may well be described as the formation of a
NAC.
O
NH2
ω-TA
NH2
O
Figure 5: A transamination equilibrium reaction where (S)‐1‐phenylethylamine and acetone is produced from acetophenone and isopropylamine, catalysed by an ω‐
transaminase (ω‐TA).
Transamination is the process in which an amine and a ketone react
to interchange their functional groups, producing the corresponding
ketone and amine (Figure 5). In a living cell an amino acid can in
this way be formed from the corresponding keto acid, by PLPdependent transaminase enzymes.5 In this case the transaminase is
labelled by the Greek letter α, having an α-amino acids and α-keto
acids as substrates and products (not the reaction depicted in
Figure 5).
Transaminases which are able to accept aliphatic ketones and
amines as their substrates, i.e. not only keto acids and amino acids,
are labelled with the Greek letter omega (ω-transaminase, EC
11
H
Lys
O
NH
O
P
O
O
NH2
OH
O
O
P
O
O
O
Lys
OH
N
H
Planar quinonoid
NH2
NH
NH H NH
2
O
P
O
O
N
H
OH
Lys
N
H
O
O
O
NH2
O
NH
O
P
O
O
O
NH2
OH
NH
O
P
O
O
O
Lys
N
H
OH NH2
H2O
O
O
O
O
O
O
Planar quinonoid
NH H
O
P
O
O
OH
N
H
Lys
N
H
H2O
O
NH2
Lys
O
O
H
NH
P
O
O
OH
N
H
O
O
NH2
Lys
O
O
PMP
P
O
O
NH2
OH NH3
N
H
Lys
Figure 6: Proposed reaction mechanism of an (S)‐selective ω‐transaminase. PLP is, in the holo enzyme, covalently bound to a lysine residue (top left) as an internal aldimine. An amino donor, here (S)‐1‐phenylethylamine, reacts with the internal aldimine to form PMP and a keto compound (here acetophenone). Then, a keto substrate, here pyruvate, reacts with PMP to form alanine. This equilibrium reaction in this case strongly favours the products. Adapted from the mechanism deduced for aspartate α‐transaminase.20,46
2.6.1.18).48 Figure 6 depicts the putative reaction mechanism,
adapted from the mechanism of aspartate α-transaminase.20,46
In the proposed mechanism, the amino donor substrate forms a
Schiff base with PLP and lowers the pKa of a proton which thereby
may be abstracted to form a planar quinonoid moiety. Thereafter
electron and proton rearrangements and a hydrolysis reaction yields
the corresponding ketone and PMP (Figure 6). When another ketone
enters the active site essentially the same reaction steps in reverse
will result in the reformation of the holo enzyme and the
corresponding amine. The enantiospecificity is presumably a result
of unfavoured coordination of one enantiomer in a catalytically
12
productive conformation, due to lack of space. In the ω-transaminase from Vibrio fluvialis the active site can be modelled as
exhibiting a large and a small pocket in which the groups bound to
the nitrogen binding carbon on the substrate are positioned after
reaction with PLP/PMP.49 Since the proton is abstracted by the
catalytic lysine, which also binds PLP in the holo enzyme, positioned
on one side of the Schiff base structure one distinct conformation
will be favoured.49 The same rational is assumed to apply for the ωtransaminase from Chromobacterium violaceum and Arthrobacter
citreus which are used in this work.
Amines are found in living cells. Dopamine, epinephrine (adrenaline)
and serotonin are examples of amines present as signal substances
in mammals. Amines are also common motifs in pharmaceuticals.
Synthesis of a wide range of enantiopure amines are of great interest
for the pharmaceutical industry.50-52 The use of ω-transaminases for
this purpose has been shown to be feasible and is an attractive
option since the enzymes have been found to display substrate
promiscuity and high enantiospecificity.48,50-67 Still, effective
synthesis requires engineering for addressing inherent obstacles in
the enzymes function. The equilibrium reaction results in low
product yield. Also, many substrates and products inhibit the
enzyme which practically halts the reaction at high concentrations.
These are unwanted limiting factors in large scale synthesis.
ω-Transaminase reactions can be categorised by distinguishing
between stereoselective synthesis and kinetic resolution reactions
(see Figure 7). The latter is referring to a process in which the
racemate of an amine is used. One enantiomer is removed by the
enantiospecific transaminase leaving the other, which can be
extracted in enantiopure form. In this case the maximum yield is
fifty percent. In synthesis mode the enantiopure amine is
synthesised from the corresponding prochiral ketone. This reaction
can in theory reach a yield of one hundred percent if the equilibrium
is displaced; in many examples of stereoselective synthesis reactions
the equilibrium favours the substrates.48,61
The impediment of the transamination reaction by substrate and
product inhibition has been tackled by means of directed evolution
with some success.64,68,69 Equilibrium displacement has been done
13
A kinetic resolution NH3
B stereoselective synthesis O
O
NH2
ω-TA
NH2
ω-TA
O
NH2
NH2
O
Figure 7: Examples of two types of reactions with an (S)‐selective ω‐transaminase (ω‐TA). In A, a reaction defined as performed in resolution mode to obtain the (R)‐
enantiomer with a possible theoretical yield of 50%, and in B, a reaction in synthesis mode to obtain the (S)‐enantiomer with a 100% possible theoretical yield if the equilibrium can be displaced. by removal of the products or large excess of amino donor.48,66,70-75
When using isopropylamine, a cost efficient chemical, as amino
donor acetone is produced beside the enantiopure amine. Acetone is
volatile and can be removed by distillation.71 But, in larger scale
distillation is not always feasible since it requires efficient mixing
and heating; also, the distillation procedure will result in
evaporation of isopropylamine to a varying degree depending on pH
(analogous to 2-butylamine65). Raising the temperature also requires
a heat stable enzyme.
14
2 Aims and Scope
The overall aim of this work was the development of methods,
creation of improved enzyme variants and gathering of information;
to facilitate the use of ω-transaminases for chemical synthesis of
enantiopure amines. Synthesis thereof with classical methods has
limitations.52 Development of ω-transaminase for biocatalysis
applications has therefore become attractive. Nevertheless, the ωtransaminase reaction has, from a biocatalytic perspective, inherent
boundaries with opportunity for engineering and method development. The main obstacles targeted here can be summarised as
follows:
•
•
•
•
•
The equilibrium reaction results in poor product yields.
There is a lack of R-selective homologues or variants.
The specificity and enantiospecificity for a variety of desired
substrates are insufficient.
Correct kinetic comparisons of homologues and variants are
impossible due to a lack of an active site titration method.
Only a small number of three-dimensional structures have
been published, which limits the possibilities for rational
redesign.
At present there are examples of successful biocatalysis with ωtransaminases where the said boundaries have been broken by
reaction and enzyme engineering. Still, additions to this field are
indeed needed; issues of importance were here selected and
resolved. ω-Transaminase enzyme from Chromobacterium violaceum
(Cv-ωTA), and from Arthrobacter citreus in one case, was used.
An alternative solution for displacing the equilibrium in
stereoselective synthesis was needed after identifying isopropylamine as a cost efficient amino donor. An enzymatic cascade
reaction where enzyme selectivity was used to form an equilibrium
displacement system was investigated.
In a rational redesign approach, creation of variants with increased
activity, shifted enantiopreference and increased enantiospecificity
were pursued. The goal, apart from creation of enzyme variants
15
suitable for future synthesis applications, was to find key residues
in the active site.
To correctly assess and compare the activity of the engineered
enzyme variants a method for quantifying the amount of active sites
is required. Therefore the establishment of such a method was
considered important and part of the aim.
Since no crystal structure of the enzyme was available a homology
model was first made on which the engineering work was based. At
a later stage a crystal structure was included in the scope of the
project after viewing the homology model as insufficient for
describing experimental findings.
16
3 Active Site Quantification of Cv-ωTA
(Paper I)
Active sites can be titrated with e.g. a suicide inhibitor,76 the
purpose is to determine the actual amount of active enzyme. The
misfolded or for other reasons inactive enzyme fraction in a given
cultivation batch may vary. A given preparation may also contain
proteins and other contaminants which will influence concentration
determination with total protein quantification methods. Once the
molar amount of active sites has been determined the turnover
number (number of converted substrate molecules per time unit and
active site, usually given in units of s-1) for a given reaction can be
calculated. To accurately compare homologous enzymes, or enzyme
variants, the turnover number is required. If the specific activity (i.e.
the activity of total enzyme or total protein) is instead used for the
comparisons the result may be greatly misleading since the true
enzyme amount is unknown.13
PLP-dependent enzymes, including some transaminases, have
shown to be irreversibly inhibited by D-cycloserine and
gabaculine,77,78 which therefore may possibly serve as active site
titrants for Cv-ωTA. Using a derivative of PLP, namely 1deazapyridoxal-5'-phosphate, proved to yield a stable aldimine in an
aspartate transaminase.79 The Cv-ωTA was partially inhibited by
gabaculine, shown by Ward and coworkers.80 Instead of testing the
putative usefulness of inhibitors the strategy in this work was to use
a reaction which was known to be catalyzed by the enzyme for active
site quantification, not titration.
For the Cv-ωTA a reaction which was virtually irreversible was
selected; the half transamination reaction of 1-phenylethylamine to
form acetophenone (Figure 8). The hypothesis, after studying the
proposed reaction mechanism (see section 1.5, Figure 6), was that
the Enz-PLP complex will be converted to Enz:PMP by an amino
donor, but not be regenerated to a significant amount, if the formed
amino acceptor was a poor substrate or thermodynamically
favoured. When employing 1-phenylethylamine as the sole substrate
(without an amino acceptor) the formation of acetophenone was
observed together with the consumption of Enz-PLP. An excess of
17
NH2
H2O
O
PLP
PMP
Consumed (S)‐1‐PEA (mM)
Figure 8: The half transamination reaction of 1‐phenylethylamine.
0.03
0.025
0.02
0.015
y = 0.81x + 0.00005
R² = 0.99
0.01
0.005
0
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
[Enz] mM
Figure 9: The amount of consumed (S)‐1‐phenylethylamine ((S)‐1‐PEA) versus the total enzyme amount in half transamination reactions with Cv‐ωTA.
1-phenylethylamine was used with different enzyme amounts. The
amount of consumed substrate could be plotted versus the enzyme
amount to produce a linear relationship which coincided with origo
(Figure 9). The reaction includes the consumption of a water
molecule which, as the reaction is performed in aqueous buffer, will
greatly shift the equilibrium towards products.
According to Figure 9 the molar amount of active sites can be
determined by the consumption of consumed substrate. The active
fraction can be deduced from the slope of the linear regression, in
this case 81%. The consumed amount of (S)-1-phenylethylamine
was quantified by HPLC. Since the enantiospecificity for this
substrate was high, E=150 (S), the racemic substrate was used and
the peak corresponding to the (R)-enantiomer was used as internal
standard.
The observation that the Enz-PLP complex was spectrophotometrically detectable at 395 nm (yellow colour), while the Enz:PMP
complex was not, allowed further simplification of the measurement
method. By correlating the absorbance at 395 nm, where a
18
Abs 395 nm 0.25
0.2
0.15
y = 8.1x + 0.0021
R² = 0.99
0.1
0.05
0
0
0.005
0.01
0.015
0.02
0.025
0.03
Consumed (S)‐1‐PEA (mM) Figure 10: The absorbance of the holo form of Cv‐ωTA at 395 nm versus the amount of consumed (S)‐1‐PEA in a half transamination reaction.
maximum was found, to the amount of consumed substrate a direct
measurement method was made. The method requires that all active
enzyme molecules are in Enz-PLP form when the absorbance is
recorded, and the assumption that all Enz-PLP is active enzyme.
Figure 10 shows the absorbance at 395 nm versus the amount of
consumed substrate, the regressed line can be said to pass through
origo. The graph shows that the active enzyme amount can be
directly quantified by absorbance with an apparent extinction
coefficient of 8.1 mM-1cm-1, with given assumptions.
The enzyme was incubated with an excess of PLP after purification
(by IMAC) and subsequent desalting to ensure that the enzyme was
in the holo form. To rule out the putative presence of Enz:PMP
emanating from the cultivation an effective amino acceptor such as
pyruvate could be added to the incubation with PLP. This would
purge the presence of PMP, leaving only Enz-PLP. In this case the
enzyme was treated with a high excess of PLP which also binds
covalently, the presence of Enz:PMP is therefore unlikely. Still, if
present the Enz:PMP may be active enzyme which is not detected in
the method described here. Therefore the method should include the
use of pyruvate. Although the method was not tested with
homologous enzymes it is seemingly feasible for all ωtransaminases, given that the reaction is virtually irreversible in all
cases and an equal amount of PLP to enzyme can be ensured.
19
During the assay design a few noteworthy characteristics of the
enzyme were found. Phosphate buffer is not a good choice since its
presence results in poor activity and instability of the enzyme.
Phosphate may compete with PLP, which binds to the enzyme with a
phosphate group, for the same binding site. It was also found that
excess PLP was detrimental to the initial rate, i.e. the presence of
more PLP than enzyme was unfavourable (a graph is presented in
section 5.2, Figure 16). A similar effect was found with an ωtransaminase from Arthrobacter citreus (see section 4).
When incubated with PLP a time dependent activation was observed,
the initial reaction rate increased by 129% without additional
addition of PLP. This effect may be a result of dimerisation for
formation of active enzyme, if the IMAC-purification resulted in
monomerisation upon binding to the used cobalt(II) containing resin.
Or, and perhaps more likely, the result is a consequence of
structural rearrangements, described in section 6.
20
4 Equilibrium Displacement of ω-Transaminase Reactions with Isopropylamine
as Amino Donor (Paper II)
Since the ω-transaminases catalyze an equilibrium reaction
complete conversion is inherently impossible without equilibrium
displacement. Virtually complete reactions can be achieved in
kinetic resolution reactions with an excess of amino acceptor; or a
catalytic amount of pyruvate as amino acceptor if combined with an
amino acid oxidase,66 thus regenerating the pyruvate from formed
alanine. Stereoselective synthesis reactions have generally proven to
reach meagre conversions although excess amino donor is used, and
are therefore subject to reaction engineering. Theoretically it is
possible to reach a hundred percent yield with equilibrium
displacement. Enzymatic cascade reactions have been published
where alanine was the amino donor; the formed pyruvate was
further reacted by lactate dehydrogenase48,71,72,81,82 or pyruvate
decarboxylase.70 When using isopropylamine as amino donor the
equilibrium can be displaced by distillation of the formed acetone,71
but this method is not practical in large scale as mentioned in
section 1.5. Employment of two-phase,61,81,83 or even three-phase
(solvent bridge84) systems have proven useful for product removal,
thus limiting product inhibition; as well as use of ion-exchange
resin,85 and continuous extraction procedures.63
As a model substance 1-Phenylethylamine was chosen, being a
small chiral aromatic primary amine. The prochiral ketone
acetophenone was therefore used to produce (S)-1-phenylethylamine; thereafter chosen derivatives thereof were produced
from their corresponding amines. Isopropylamine was the chosen
amino donor, a cost efficient industrial chemical.
An alternative method from distillation for removing acetone in a
transamination reaction in synthesis mode with isopropylamine as
donor was explored; the acetone was converted to isopropanol with
the enzyme yeast alcohol dehydrogenase (Saccharomyces cerevisiae,
YADH). This alcohol dehydrogenase is selective for small alcohols
and ketones.86 The amine acceptor is most often a larger ketone,
hence unaffected by the presence of YADH. This is an example of
21
O
NH2
NH2
ω-TA
O
O C O
NADH,H+
YADH
FDH
NAD+
OH
-HO
O
H+
Figure 11: A transamination reaction with isopropylamine as donor and acetophenone as acceptor, catalysed by an ω‐transaminase. The equilibrium is displaced by selectively removing the formed acetone with YADH and regenerating the cofactor NADH with FDH.
utilisation of enzyme selectivity for practical use in an enzymatic
cascade reaction. The cosubstrate NADH was regenerated with
Candida boidinii formate dehydrogenase (FDH), the production of
carbon dioxide effectively drives the reaction to completion. Figure
11 shows the used reaction scheme.
The gene for an Arthrobacter citreus ω-transaminase variant69,87 was
cloned and successfully overexpressed in E. coli BL21(DE3), and
purified by IMAC. The influence of excess concentration of the
coenzyme PLP was examined; it was found that exceeding the
concentration beyond that of the enzyme was detrimental to the
reaction. Figure 12 shows the initial reaction rate versus the excess
concentration of PLP, it is clear that the rate is greatly decreased
with increasing amount of coenzyme. Since the enzyme
concentration is usually very low, in the micromolar range, this
effect is of importance. Already at millimolar concentrations PLP will
practically completely discontinue the reaction.
Other examples of inhibition by PLP have been published, e.g. of
RuBisCO,88 and of thymidylate synthase89 where a putative thiohemiacetal was formed by PLP and a cysteine residue. Although
these are related examples it is unlikely to be a similar mechanism
of inhibition as for the ω-transaminase here. Arguably, enzymes with
a lysine residues in the active site will be inhibited by PLP to some
extent if a Schiff base with PLP can be formed, regardless if they are
PLP-dependent or not. The mechanism for the inhibition of Cv-ωTA
observed here remains to be investigated.
22
Rate (U/mg)
1
0.8
0.6
0.4
0.2
0
0
100
200
300
400
500
(excess [PLP])/[Enz] Figure 12: Activity versus excess PLP, more PLP than enzyme results in lowering of the initial rate.
A balanced ratio of PLP to enzyme was obtained by dissolving an
excess of PLP in the purified enzyme solution and then changing the
buffer. Assuming that no enzyme dissociates from the PLP during
this process this provides an enzyme ready for use with the correct
amount of coenzyme. No coenzyme was thereafter added in the
reactions.
Acetophenone is a poor substrate (i.e. has a comparatively low
specificity constant) but is advantageous since it is spectrophotometrically detectable while the corresponding amine shows a
significantly lower extinction coefficient.90 A continuous spectrophotometric measurement verified the equilibrium displacement
system. Figure 13 shows a comparison of the displaced versus the
non-displaced reaction as the consumption of acetophenone over
time. The displaced reaction continued towards completion while the
non-displaced virtually stopped. Here the initial concentration of
acetophenone was low, only 1.8 mM. This concentration was chosen
to keep the starting absorbance at a spectrophotometrically
measurable level and to have a practical conversion point for
comparison. Even with a large excess of amino donor, 564 mM of
isopropylamine was used, the conversion (without displacement)
only reached 82%. The amount of formed product, (S)-1phenylethylamine, and the enantiomeric excess was confirmed by
HPLC. Indeed, the displaced reaction had virtually reached
completion and the product amine was enantiomerically pure.
23
[acetophenone] mM
2
Equilibrium reaction
Displaced reaction
1
0
0
6
12
18
Time (h)
Figure 13: Comparison of a displaced and a non‐displaced transamination reaction, followed by continuous absorbance measurement of the consumption of acetophenone.
Table 1: Results from stereoselective synthesis reactions performed with
a variant of A. citreus ω‐transaminase with isopropylamine as amino
donor (564 mM), with and without the displacement system. Product amine ee (%) Conversion (%) no displacement NH2
Conversion (%) with displacement >99.9 82 a >99 a >99.9 63 b >99 b >99.9 89 c >99 c >99.9 68 c >99 c NH2
O2N
NH2
NH2
HO
OH
a
1.8 mM acetophenone. Conversions were calculated based on formed product. b
20 mM 4‐nitroacetophenone (two phase system as saturation is reached at ~2 mM). Conversions were calculated based on formed product. c
20 mM ketone substrate. Conversions were calculated based on consumed substrate. The displacement system was then explored for production of
derivatives of (S)-1-phenylethylamine. Table 1 summarises the
results; with 20 mM substrate concentration the displaced reactions
also reached completion. No workup was performed, conversions
were measured by HPLC.
24
PLP
Enz
NH
NH2
H2O
Enz-PLP
O
Enz PMP
NH2
PLP
Enz
NH
O
H2O
Figure 14: Schematic representation of the ω‐transaminase catalysed reaction as half transamination reactions, of isopropylamine/acetone (top) and acetophenone/(S)‐1‐phenylethylamine (bottom). Higher product concentrations (20 mM was achieved here) were not
feasible. It was later observed that the specificity constant for the
chiral amino product was many times higher than for
isopropylamine, similar to published results with homologous
enzymes48,62,81 and the results with Cv-ωTA in section 5.2. It is
therefore unlikely that the product concentration limitation is a
result of an unfavourable equilibrium, i.e. a thermodynamic effect,
since displacement was in fact used. Rather, it is more likely a
kinetic effect due to the specificity of the enzyme; although the
thermodynamic equilibrium distribution was not measured.
Figure 14 shows a simplified scheme of the reaction mechanism
with the half transamination reactions separated. If the specificity
constant for the formed product, (S)-1-phenylethylamine, is higher
than for the amino donor, isopropylamine, the enzyme will
practically seize to catalyse the half reaction of isopropylamine as
the concentration of (S)-1-phenylethylamine increases. The virtual
discontinuation of the reaction is a result of a competing substrate
situation arising from the formation of the chiral product which can
function as an amino donor, in which case the isopropylamine will
be disfavoured. At a given point an excess of isopropylamine will be
insufficient for upholding continued production of the product
amine due to the competing substrate situation, which will be
observed as the termination of product formation. The bottom half of
25
the reaction scheme in Figure 14 may still be catalysed but will not
be detected. Displacement of the equilibrium alone will not resolve
this issue. Higher product amounts may be achieved by continuous
removal thereof, which would enable the enzyme to react with
isopropylamine as amino donor.
26
5 Rational Redesign of Cv-ωTA (Paper
III and IV)
Due to the lack of crystal structures protein engineering of ωtransaminases has to the most part been based on directed
evolution approaches, where the activity, stability and enantioselectivity has been targeted.64,69,74,91
In a rational redesign approach, however, a tryptophane residue was
targeted in the ω-transaminase from Vibrio fluvialis.92 Kim and coworkers state that when changed to a glycine residue the enzyme
displays higher activity and enantioselectivity for a number of
aliphatic amines due to increased space in the active site. The same
rational was applied for Caulobacter crescentus ω-transaminase93. In
these cases homology models were applied.
Hughes and co-workers also applied a homology model on an
enzyme variant from Arthrobacter spp. where a semi-rational
substrate walking approach yielded an enzyme suitable for synthesis
of sitagliptin.60 This is an example of application of an ωtransaminase for the industrial production of a pharmaceutical.
(S)-enantiopreference appears to be most common in nature; still, a
number of ω-transaminases with (R)-preference were found by
Bornscheuer and co-workers through computational search
methods.94 High yield stereoselective synthesis of (R)-amines have
been published by Kroutil and co-workers,95 and by Bornscheuer
and coworkers.96 Although rational redesign has been used to
achieve a substrate dependent shift of enantiopreference from (S) to
(R),97 it still is a challenging engineering task which may lead to
increased understanding of the enzyme when successfully achieved.
Other PLP-enzymes have been extensively studied and a number of
successful examples of rational redesign are published. E.g. alanine
racemase from Geobacillus stearothermophilus, as described in
section 1.5, accepted serine as a substrate when more space was
made in the active site which enabled a catalytically productive
conformation.47
27
A
H'318
B
Pro-(S)
G'319
G'319
F'320
T'321
A231
O
P
O
O
OH
F'88
F'320
T'321
NH
O
Pro-(R)
H'318
F'88
A231
NH
O
P
O
O
O
W60
OH
N
H
N
H
O
W60
O
O
O
D259
D259
Figure 15: Schematic representation of the active site, deduced by a homology structure, with planar quinonoid structures resulting from the predicted reaction with (S)‐1‐phenylethylamine in A and (R)‐1‐phenylethylamine in B.
The Cv-ωTA was found to have good enantioselectivity for e.g. 1phenylethylamine; but for a variety of substrates the E-values were
not high enough for biocatalytic application. The possibility of
creating a general enzyme, i.e. good activity and high E-value for a
vast number of substrates, is unlikely. Such an enzyme would need
to be able to accommodate large substrates, i.e. large groups on
either side of the amino/keto group, and still bind small substrates
in only one defined conformation. This is unreasonable since smaller
substrates would be able to bind in a catalytically productive
manner in mirrored conformations in an enlarged active site which
would result in poor enantiospecificity, as also mentioned by Kroutil
and co-workers.98
Here the goal was to increase the enantiospecificity and activity for
chosen substrates by rational redesign, and in doing so, finding key
amino acid residues in the active site for future engineering efforts.
Also, the possibility of shifting the enantiopreference was explored.
A crystal structure was not available. Instead a homology model was
constructed in accordance with a previously published method.97 A
transaminase from Silicibacter pomeroyi with available crystal
structure with sequence similarity of 51% was used as a fold
template for Cv-ωTA. The rational redesign was then based on this
homology model, at a later stage a crystal structure was made which
is described in section 6.
28
5.1 Improved enantioselectivity and reversed
enantiomeric preference (Paper III)
A schematic visualization of the active site is presented in Figure 15
with planar pro-(S) and pro-(R) quinonoid structures. The engineering approach was based on the assumption that binding of
these planar quinonoid structures are essential for the resulting
enantiospecificity, i.e. the difference of stability of the enzyme-planar
quinonoid complex for the pro-(S) versus the pro-(R) quinonoid
structure is a determinant for the difference in activation energy of
the enantiomers in the transamination reaction.
Several mutants were made (unpublished results) based on the
schematic picture in Figure 15, with the straightforward assumption
that the stability of the binding of the quinonoid structures from
reactions with (S)- or (R)-forms of unnatural substrates is to the
most part dependent on space in the active site. Hypothetically, if
the enantioselectivity is a result of unfavoured coordination of e.g.
the pro-(R) quinonoid, thereby favoring the pro-(S), the
enantioselectivity may be reversed if the amino acid residues are
changed and more space is made for the pro-(R) and less for the pro(S). In silico docking experiments were performed to guide the choice
of mutation points. The structures in Figure 15 are the reaction
intermediates formed after abstraction of the proton from the Schiff
base. The probability for proton abstraction, which is plausibly a
rate determining step, is in this model assumed to be proportional to
the coordination for the resulting structures. This model is therefore
seen as a simplified guide, not an attempt to predict the actual
activation energies for reactions of the enantiomers.
One variant made according to this hypothesis was a success, with a
substrate dependent shift of enantiopreference. Table 2 displays Evalues for the chosen substrates in five chosen variants.
A variant with two amino acid residue substitutions, phenylalanine
88 to alanine and alanine 231 to phenylalanine, showed a switch of
enantiopreference for 2-aminotetraline with good enantiospecificity.
Practically, the variant consists of a replacement of a phenyl group.
For 1-aminotetraline this variant also showed a shifted enantiopreference, but with a low E-value. For 1-phenylethylamine the
29
Table 2: E‐values for chosen substrates in Cv‐ωTA wild type and variants, with specified enantiopreference.* NH2
Enzyme wild type Trp60Ala Trp60Cys Phe88Ala Ala231Phe Phe88Ala, Ala231Phe NH2
NH2
150 1.5 340 7.1 15 6.3 (S) (S) (S) 6.7 1.0 60 (S) ‐ (S) (S) 1.2 (R) (S) 6.0 (S) (S) 1.9 (R) 3.9 5.3 59 no reaction 1.0 63 (S) (S) (S) ‐ ‐ (R) *Reactions run with pyruvate (1 mM) as amino acceptor.
enantiopreference was not shifted, but a lowered E-value was
observed. This substrate dependent shift of enantiopreference
remains to be explored for other substrates. It is possible that (R)selectivity will be observed for aromatic amines with one methylene
or more to separate the amino moiety from the phenyl group, such
as amphetamine and derivatives thereof. According to the
hypothesis (described in section 1.5) of the enantioselectivity largely
being dependent of a large and a small pocket in the active site, the
enzyme would have higher enantioselectivity for amines with larger
groups. This is consistent with the variant having higher
enantioselectivity for 2-aminotetraline than 1-aminotetraline. Kinetic
resolution or stereoselective synthesis of (S)- and (R)-2aminotetraline is interesting from a pharmacological standpoint. The
substances and their derivatives are central nervous system (CNS)active drug compounds.99 1-phenylethylamine is here used as a
model substrate since it is a small aromatic chiral amine. However,
it may not be a satisfactory representative for the enantiospecificity;
there is a possibility that the corresponding planar quinonoid
structure displays rigidity for rotation of the phenyl ring due to the
π-electron system.
To increase the enantiospecificity of the (S)-selective Cv-ωTA the
strategy was, analogous to the rational for shifting enantiopreference, to favour coordination of the pro-(S) quinonoid. The
tryptophan residue on position 60 was targeted since the homology
model predicted this amino acid residue to significantly influence
the space in the active site for the phenyl group from (S)-130
phenylethylamine in the quinonoid; if changed for a smaller residue
the pro-(S) conformation should be more favoured than in the wild
type. Contradictory to this basic hypothesis was the finding of the
variant with an alanine residue on this position (Trp60Ala) to have
lower enantioselectivity, as well as lower activity. The Trp60Ala was
also an unstable enzyme which made experimental procedures
complicated.
Instead, when a cysteine residue was introduced on position 60 a
stable and effective enzyme was obtained. The choice of cystein
originated from comparison with a literature variant found by
directed evolution of Arthrobacter citreus ω-transaminase;69,87 this
variant has a cysteine residue in the corresponding position which
could be identified with the homology model. Introduction of
cysteine residues is not otherwise a common choice since it is a
reactive species.
As displayed in Table 2 the increase of the E-values for the tested
substrates of the Trp60Cys variant are significant. The E-value of
this variant for 1-phenylethylamine, 1-aminotetraline and 2aminotetraline are high enough for use in kinetic resolution
reactions, whereas the E-value for the wild type is only adequate for
1-phenylethylamine.
31
5.2 Increased specificity (Paper IV)
The increased E-values of the variant Trp60Cys presented in section
5.1 was a result of increased specificity for the (S)-enantiomer, not a
lowering for the (R). A variant with a lowered catalytic activity may
have an improved E-value if the specificity for the slower reacting
enantiomer is fractionally more decreased than the specificity for the
faster. This is an undesired result which was not the case for the
variant Trp60Cys. The variant showed increased reaction rates for
the (S)-enantiomers compared to wild type.
To further explore the function of the variant specificity constants
for (S)-1-phenylethylamine, isopropylamine and a number of 4’substituted acetophenones were determined. The active site
quantification method (section 3) was used and turnover numbers
could be calculated. As described in section 1.3, the apparent
(pseudo one-substrate) specificity constants can be regarded as the
true constants in the case of an ordered ping-pong bi-bi reaction,
which applies to the ω-transaminase reaction.
The pH optimum was measured to 7.0 for Trp60Cys and 8.3 for the
wild type. A study of the coenzyme dependence showed that the wild
type displayed lowered rates with excess PLP whereas the variant
showed enhanced rates and was dependent on excess PLP to reach
maximum rate, see Figure 16. The reactions with the variant and
the wild type described below were performed at the found optimum
conditions, i.e. not equal pH and coenzyme concentrations. HEPES
buffer and 37 °C was used in both cases. As described in section 3,
phosphate inhibited and destabilised the enzymes, presumably due
to competition with the phosphate group of PLP.
Kinetic constants were experimentally determined for (S)-1phenylethylamine by spectrophotometrically following the formation
of acetophenone,90 and for isopropylamine by quantifying the
amount of formed alanine after five minutes with ninhydrin.
Isopropylamine does not produce a colour reaction with
ninhydrin,100 whereas alanine is readily detectable. A fixed
concentration of pyruvate (1.0 mM) as amino acceptor was used.
The values are shown in Table 3. The specificity constant for (S)-1phenylethylamine was increased 29-fold for the variant Trp60Cys,
32
activity (%)
100
80
wild type
60
Trp60Cys
40
20
0
0
2
4
6
8
(excess [PLP])/[Enz]
Figure 16: The effect of excess PLP on the initial rate of Cv‐ωTA wild type (blue dots) and variant Trp60Cys (red diamonds). The wild type enzyme was inhibited while the variant did not reach maximum rate below a two‐fold excess of the coenzyme. Upon studying the continuation of the reaction, the variant required 3‐
fold or more excess of coenzyme in order to maintain the observed initial rate.
Table 3: Kinetic constants for (S)‐1‐phenylethylamine and isopropylamine for Cv‐ωTA wild type and variant Trp60Cys.* NH2
M
(s‐1∙mM‐1) Cv‐ωTA wild type Cv‐ωTA Trp60Cys 0.68 20 NH2
M
(s‐1∙mM‐1) M (mM) (s‐1) 0.20±0.004 73 15±0.3 0.024±0.002 190 4.6±0.4 *Reactions performed with pyruvate (1 mM) as amino acceptor.
compared to the wild type. Isopropylamine, on the other hand, was a
poorer substrate for the variant in terms of turnover number and
affinity. The KM value for isopropylamine had increased more than
two-fold and the turnover number was more than three times lower.
By allowing the chosen 4’-substituted acetophenones to compete for
the enzyme, with isopropylamine as amino donor (50 mM for the
wild type and 200 mM for the variant), relative specificity constants
were obtained. Three ketones (0.2 mM each) were employed
33
Table 4: Specificity constants for chosen 4’‐substituted acetophenones for Cv‐ωTA wild type and variant Trp60Cys. O
X
M
X: NO2 Cl Br CN F H Me OH MeO (s‐1∙mM‐1) Cv‐ωTA wild type 0.030 0.029 0.028 0.026 0.020 0.0082a 0.0050 0.0024 b
n.d. Cv‐ωTA Trp60Cys 0.16 n.d.c 0.039 0.10 c
n.d. 0.034a 0.024 0.013 0.011 a
Experimentally determined values from which the remaining values were calculated from their relative values, IPA (50 mM for wild type, 200 mM for Trp60Cys) was used as amino donor. b
Not determined, no observed reaction (<0.0001). c
Not determined, background reaction higher than the enzyme catalysed reaction.
simultaneously in each reaction; inter-comparison was done by
common ketones to link the reactions. The rates were obtained by
studying consumption over time by HPLC. This is an effective and
precise way of obtaining the relative specificity constants. As stated
in section 1.3, the ratio of rates for competing substrates simplifies
to the ratio of specificity constants. Dilution errors for enzyme
concentration and time measurement errors cancel out from the
division of the equations. The rate for acetophenone was then
spectrophotometrically determined90 and used for calculating the
remaining rates from their relative values. In Table 4 the chosen 4’substituted acetophenones are listed with their corresponding
specificity constants, for the variant and the wild type enzyme.
An average 4-fold increase of the specificity constant for the 4’substituted acetophenones was observed for the variant compared to
the wild type. Overall, an electron withdrawing group as 4’substituent increased the specificity constant while electron
donating groups led to a decrease. This observation warranted a
study of what effects governed the observed reactivity of the
acetophenone derivatives, a parameterisation approach was chosen.
34
For reactions of benzoic acid, and derivatives thereof, the SwainLupton equation101-103 can often be used for finding the electronic
effect of substituents on the reaction rate, in terms of the field and
resonance contributions separately. The Swain-Lupton equation is a
refinement of the Hammett equation,104 which does not distinguish
between field and resonance effects; an empirical constant for a
given substituent is derived based on the difference of pKa of
substituted benzoic acids. The Swain-Lupton equation assumes field
(and inductive) effects to be independent of resonance. By linearly
combining the terms the equation gives:
(eq. 9)
where k is the rate constant for a substituted species, k0 is the rate
constant for the unsubstituted species, f and r are reaction specific
constants, and F and R are deduced values for each substituent
which are shown in Table 5.
The specificity constants in Table 4 were treated as first-order rate
constants for parameterization and least squares-fitting to the
Swain-Lupton equation. In this model the ordered ping-pong bi-bi
two-substrate enzyme reaction has been drastically simplified to a
first order reaction; the enzyme and amino donor are treated as
constants. For determination of the field and resonance effects
alone, and for comparison of the variant to the wild type, the model
is applicable. The reaction conditions differed for wild type and
variant. The pH, coenzyme concentration and amino donor
concentration were optimised for each enzyme. There is a possibility
of the results being biased or condition specific, but the alternative
would be to evaluate the field and resonance effects at suboptimal
conditions. The comparison made above regarding the specificity
constants in Table 4 are indeed done in absolute terms, but from a
practical perspective. In the parameterisation comparisons of the
fractional contribution of field and resonance for each enzyme are
made, not the absolute values. The Swain-Lupton plots are shown in
Figure 17 together with the fitted equations.
35
A Cv‐ωTA wild type 0.8
0.6
NO2‐
Cl‐
y = 1.0001x ‐ 9E‐05
R² = 0.999
0.4
Br‐
CN‐
log (k/k0)
F‐
‐1
0.2
0
‐0.5
0
0.5
1
1.5
‐0.2
Me‐
log
0.90
0.50
‐0.4
OH‐
‐0.6
Swain‐Lupton
B Cv‐ωTA Trp60Cys
0.8
NO2‐
0.6
log (k/k0)
y = 1.0002x ‐ 0.0014
R² = 0.9839
CN‐
0.4
0.2
Br‐
0
‐1
‐0.5
Me‐
0
0.5
1
‐0.2
MeO‐
‐0.4
OH‐
log
0.28
0.34
‐0.6
Swain‐Lupton
Figure 17: Swain‐Lupton plots for reactions of chosen 4’‐substituted acetophenones from the rate constants listed in Table 4, for Cv‐ωTA wild type in A and variant Trp60Cys in B. The 4’‐substitution of the acetophenones are shown in the graph. The values of the coefficients f and r were calculated to 0.90 and 0.50 for the wild type, 0.28 and 0.34 for Trp60Cys. The contribution of resonance is 36% in the wild type and 55% in the variant. 36
Table 5: Swain‐Lupton parameters101‐103
for chosen 4’‐substituents. NO2 CN F Br Cl H Me OH MeO Field parameter (F) 1.0 0.9 0.74 0.72 0.72 0 ‐0.01 0.46 0.54 Resonance parameter (R) 1.0 0.71 ‐0.6 ‐0.18 ‐0.24 0 ‐0.41 ‐1.89 ‐1.68 For the wild type the fitting was excellent except for the reactions of
4’-cyano- and 4’-nitro-acetophenone, in the variant the fitting was
good over the full range of chosen reactions. This can be interpreted
as the catalytic contribution by the variant being equal for all these
substrates, the rate differences can be attributed to the activation or
deactivation by the substrates alone. In the wild type the same can
be said except for the cyano and nitro substituents for which the
Swain-Lupton predicted reaction rates are faster than the experimental values. From this point of view the variant may be seen as a
more general catalyst. The contribution of resonance effects are
governing the specificity in the variant (55%), compared to the wild
type (36%) where field and inductive effects appear to be dominant.
The variant is not expected to have altered electronic, coenzyme and
pH dependence without a change in the catalytic mechanism; e.g. a
shift of rate determining step. It is conceivable that the introduced
cysteine residue, a reactive species, can function as a Lewis base
and thereby significantly influence the reaction. The thiol group may
provide a field effect which lessens the importance of the effect by
the substituent. In cannot, however, provide a resonance
contribution if is not bound to the substrate-PLP complex.
37
The reason for the lowered E-value of the Trp60Ala variant (section
5, Table 2) is possibly a result of destabilization; it is possible that
the specificity constant for the (S)-enantiomer was lowered by a
larger fraction than for the (R). The low expression yield is consistent
with a destabilized enzyme, possibly the enzyme produced inclusion
bodies. The Trp60Ala variant was not considered a subject for
deeper investigation since the practical applications were limited
and the low activity made testing complicated.
38
6 Structure of Cv-ωTA (Paper V)
The discovery of Cv-ωTA as an ω-transaminase was done by
sequence identification with the ω-transaminase from Vibrio fluvialis
as template, by Ward and co-workers.105 PLP-dependent enzymes
are classified in fold types (I-V) by their tertiary structure and into
subgroups (I-IV) by their sequence identity.106-108 Cv-ωTA belongs to
fold type I and subgroup III. Aspartate transaminase also belongs to
fold type I, but subgroup I. Cv-ωTA is (S)-selective (L-amino acid) and
the catalysed reaction is assumed to follow the same reaction steps
as an aspartate transaminase;20 the sequence dissimilarity,
however, indicates that the enzyme has different structure. Crystal
structures of ω-transaminases may be a necessary basis for further
exploration.
Although the rational redesign described in section 4 was performed
with use of a homology model, a more complete structure of Cv-ωTA
was desired. In the model, coordinated water molecules were not
included and amino acid residue side chain rotational positions
could not be assured. Additionally, the homology model could not
serve as a base for a hypothesis about the experimental findings
described in section 2; when incubated with PLP the enzyme
displayed a time dependent activation. A crystal structure is the
preferred base for further engineering, notwithstanding the success
with the homology model.
There are examples of successful homology model based
engineering,60,92,93 described in section 5. Still, it is conceivable that
more precise structures of both apo and holo forms of an ωtransaminase may reveal influential functions for further
understanding and development. There are available crystal
structures of ω-transaminases; the apo enzyme from Vibrio fluvialis
by Jang and co-workers (PDB ID: 3NUI) and one monomer of the
holo enzyme from Psudomonas putida by Sasaki and co-workers
(PDB ID: 3A8U). These structures are presented without a published
structural
analysis.
Experimental
details
regarding
the
crystallisation of Cv-ωTA have been published by Littlechild and coworkers,109 the structure itself was published shortly after Paper V
(PDB ID: 4AH3).
39
X-ray crystallographic structures of both apo and holo forms of CvωTA were produced at resolutions between 1.35 and 2.4 Å, one of
the holo form (PDB ID: 4A6U), two in the apo form (PDB IDs: 4A6R
and 4A6T) and one in a mixed form (PDB ID: 4A72). Figure 18 shows
the enzyme in α-helix/β-sheet/flexible loop representation.
The enzyme crystallised as a homodimer. Each monomer had a PLPbinding site at the subunit interface. The homodimer thereby has
two active sites composed by elements from both subunits, similar
to homologous enzymes.
The structures revealed significant differences between the holo and
apo forms, also stated by Littlechild.109 This was interpreted as
structural rearrangements upon coenzyme binding. With bound PLP
the enzyme appears more ordered while the apo enzyme has flexible
parts on one side of the active site. The observed time dependant
activation may be a result of these rearrangements.
The holo enzyme crystal structure was rather similar to the
homology model. Out of the 916 Cα-positions 896 could be aligned
with an RMSD of 1.01 Å, with a seemingly identical active site apart
from a few side-chain rotamer positions. The active site of the holo
enzyme is shown in Figure 19.
The structural rearrangements upon coenzyme binding are of
significance for the form of the active site. The parts coloured in
yellow in Figure 19 are part of a structure which may be envisioned
as a lid which partly covers the active site when PLP is bound. The
reordered section consists mainly of residues 317-322 of the subunit
which is not binding PLP. The threonine residue 321 and tyrosine
residue 322 are repositioned in proximity to the internal aldimine
complex and are likely to be of significance for the coenzyme and
substrate binding. When repositioned, the coenzyme is coordinated
by more hydrogen bonds; the phosphate group of PLP appears to be
fixed and the otherwise flexible loop of residues 287-290 is
positioned so that the PLP binding lysine residue 288 is projected
into the active site. The described conformations of the apo and holo
forms of the active site region are shown in Figure 20.
40
A B Figure 18: Overall fold of Cv‐ωTA in the holo form with the large domain coloured pink, the two small domains coloured light and dark blue and the linker containing an α‐helix in grey. The monomer is depicted in A, and the native dimer in B. (Courtesy of Assoc. prof. Derek Logan, Lund University)
41
Figure 19: Graphical representation of the active site of Cv‐ωTA in the holo form, composed of elements from both subunits of the homodimer distinguishable by grey and yellow colour. Amino acid residues within 4 Å of PLP are shown in stick representation together with the PLP‐internal aldimine complex inside a semitransparent grey surface. Hydrogen bonds are depicted as dotted lines, water molecules participating in hydrogen bonding with the aldimine are shown as red spheres. (Courtesy of Assoc. prof. Derek Logan, Lund University)
42
Figure 20: Graphical representations of the active site in the apo (A) and holo (B) form with the PLP‐binding subunit in light blue, the other subunit in beige and the rearranging structures in dark red. (Courtesy of Assoc. prof. Derek Logan, Lund University)
43
7 Concluding Discussion
Evolution of enzymes has proven to be a success story for life, all
living organisms can sustain their metabolic needs by enzyme
catalysis of which the qualities remain to be fully utilised in
chemical engineering. Many enzymes are presently being utilised in
industry and academic research, but the current exploitation does
not by any means reach the full potential.
When a chemist is presented with a task of synthesising a given
chemical the available methods commonly suggest a classical
approach where organic solvents and metal catalysts rule. If the
chemist encounters a reaction step which cannot be accomplished
with this approach, an enzyme as an alternative method might be
sought after for this specific step in the process. Thereby the enzyme
is hoped to cope with, or to be adapted to, the from an
enzymologist’s perspective rough conditions. The fundamental
strategy should be to devise an entire process based on biocatalysis,
in aqueous conditions. This approach has been discussed as the
development of cascade chemo-enzymatic processes or enzyme
initiated domino reactions.40,110-112 This requires further method
development and demonstrations of successful large scale reactions.
The choice of product may play a paramount role. A compound for a
desired application may be selected within a range of suitable
products for a complete biocatalytic synthesis method, assuming
that not only one specific compound will suit the application. Today
the choices may be based on available classical synthesis methods,
which may not be beneficial in a long term perspective. Biocatalysis
is currently in a sense only competing with the reactions which are
not available or not satisfactory with other methods. In a life cycle
analysis, a calculation of the total production cost of a chemical,
where waist disposal and environmental impact is included a
biocatalytic synthesis may be the most cost efficient;113 particularly
if environmental damages are to a larger extent subject to
economical recompense enforced by legislation.
44
7.1 Reactions and engineering with ω-transaminase,
summarising discussion of Paper I-V
The reactions catalyzed by ω-transaminases readily compete with
classical methods. The enzyme can form a chiral amine from a
prochiral ketone, the amination is in itself an advantage and the
enantioselectivity has in many cases been shown to be high enough
for production of virtually enantiopure compounds.58
The described active site quantification method (Paper I) provides a
means for correct assessments and comparisons of engineered
variants and homologues. The turnover number and finding optimal
reaction conditions is many times overlooked, and arbitrary
comparisons in given conditions with an unknown active enzyme
fraction are then made. This cannot be considered a satisfactory
procedure since it stands the risk of giving greatly misleading
values. Active site quantification is also beneficial when employing
an enzyme in a chemical process to find the expected catalytic
efficiency. The method presented is a straightforward procedure; a
simple spectrophotometric measurement will yield the amount of
active sites. The measurement can be done on an enzyme batch
before employment in a reaction, no enzyme is consumed. An
alternative method for measuring the active sites, a titration
procedure, may include the use of cycloserine which is a suicide
inhibitor for many PLP-enzymes, or 1-deazapyridoxal-5′-phosphate
which can create a stable external aldimine in an aspartate
transaminase.79 A titration method may be employed to re-verify the
obtained apparent extinction coefficient, which arguably provides an
effective measurement procedure.
The use of alcohol dehydrogenase from yeast for displacing the
equilibrium of a transamination reaction with isopropylamine as the
amino donor (Paper II) was shown to be efficient, at relatively low
substrate concentrations (20 mM ketone). YADH is used for
selectively removing a compound in a cascade reaction, with
cosubstrate regeneration. Enantiopure amines were produced in
virtually complete reactions. Further development is needed to
overcome the large difference of specificity which is many orders of
magnitude lower for the amino donor (isopropylamine) than the
desired product amine, to achieve a higher total product
45
concentration. The work is an example of the possibilities that
biocatalysis can provide in terms of applications of existing
knowledge. Development of computational search tools may enable
finding of other more complex cascade reactions which otherwise
would require extensive book mining.
Rational redesign, based on a homology structure, was used to
produce ω-transaminase variants with shifted enantiopreference,
higher enantiospecificity and improved specificity for chosen
substrates than Cv-ωTA wild type (Paper III and IV). At a later stage
a crystal structure was obtained which verified the accuracy of the
used homology structure (Paper V). This successful example,
together with other published works,60,92,93 shows that it is possible
to perform rational redesign without a crystal structure on ωtransaminase by use of homology structures, although the examples
may be circumstantial. The putative reaction mechanism was based
on that of aspartic transaminase,20 another feature which was
assumed to be similar to homologous enzymes. Further engineering
on this enzyme group may benefit from continued exploration of the
reaction mechanism; especially since the mechanism possibly is
altered in the variant (Cv-ωTA Trp60Cys) with increased specificity
constants. Such experiments may be of greater value than directed
evolution approaches for further engineering by increasing the
knowledge. The results in Paper IV indicate that the reaction rate is
dependent of electronic effects which can be adapted to enhance
activity. Further engineering efforts may include a model for
alteration of the charge distribution of the active site. Reaction rates
of poor substrates may be elevated by e.g. counteracting the
deactivation shown by the electron donating 4’-substituents of
acetophenone. If a less reactive amino acid residue can be
introduced without destabilising the enzyme (Cv-ωTA Trp60Ala was
not stable) Swain-Lupton parameterisation of its reactions may show
if the enhanced specificity constants of Cv-ωTA Trp60Cys were due
to the introduced cysteine residue, and to what extent the effect
depends on increased space.
The variant Cv-ωTA Trp60Cys showed significantly increased activity
for the aromatic substrates tested compared to the wild type, but
significantly lower for isopropylamine. This may be a result of the
increased space in the active site which may allow more degrees of
46
freedom for the smaller substrate and thereby disfavouring a
catalytically productive conformation. This would imply that the
specificity for smaller substrates is inherently lowered if the enzyme
is able to accommodate larger substrates. This is contradictory to
the published data on the variant of ω-transaminase from Vibrio
fluvialis with the corresponding position changed for a glycine
residue (on which the Cv-ωTA Trp60Cys variant was partly based).92
In this case, however, the activity was measured at the same pH for
both wild type and variant, no active site quantification was
performed and the coenzyme dependence was not explored. It is
possible that the V. fluvialis enzyme variant has similar properties to
Cv-ωTA Trp60Cys regarding pH optimum and PLP dependence, and
the observed increase of activity for isopropylamine is only valid at
the given reaction conditions.
The structural rearrangements upon cofactor binding revealed by
crystal structures (Paper V) is a quality which was not elucidated by
the homology modelling, which is likely to be of importance for
further engineering. One might speculate that an unnatural
substrate may induce an open form upon reaction with the holo
enzyme, thus rendering the enzyme inactive. Time dependent
activation of the enzyme was observed when incubated with the
coenzyme. The results from the crystallization experiments indicate
that the structural rearrangements are a result of coenzyme binding,
or even hydrogen bonding to inorganic phosphate in the “phosphate
group binding cup”.114 The rearrangements can be a slow process
and may be then be observed as time dependent activation. This
hypothesis is consistent with the observed deactivation of the
enzyme by phosphate buffer (sulphate and borate as well). The
mechanism behind the change in coenzyme dependence by the
variant (Trp60Cys), which was not inhibited but activated by excess
PLP, may be elucidated by a crystal structure, and could be
important information for further engineering.
It is possible that the coenzyme binding is a sequential process in
which PLP first forms a Schiff-base with the catalytic lysine residue,
and then binds to the phosphate binding cup which induces a
structural rearrangement which fixes the enzyme in the active form.
Another possibility is that PLP first binds to the phosphate binding
cup, which then induces a structural rearrangement which favours
47
formation of the internal aldimine. In either case, if the
concentration of PLP is high this process may be inhibited by the
presence of two PLP moieties, one bound to the lysine residue and
another to the phosphate binding cup, thereby preventing the
formation of the active form. If this is the case, the mode of
coenzyme binding must be different in the variant which was not
affected by excess PLP. The apo enzyme of the variant did not show
a disulphide bridge with the introduced cystein residue, which is
located adjacent to another cystein. This was observed in a
preliminary crystallographic study.115 It is unlikely that a disulphide
bond should be present in the holo form since the apo enzyme had
previously been incubated with PLP, in which case the enzyme
would have undergone oxidation subsequent to coenzyme
dissociation.
7.2 Future prospects
This section discusses ideas for possible continuation of the present
investigation.
7.2.1 Dynamic kinetic resolution of an amine
A lipase, such as Candida antarctica lipase B (CalB), can
enantioselectively acylate an amine in an organic solvent.116 If an
amine can be racemised effectively, combined with enantioselective
acylation of either enantiomer, the reactions would result in an
enantiopure amide. This has been shown by Bäckvall et al. for 1phenylethylamine with racemisation by a ruthenium catalyst and
acylation with CalB.117
Amine racemisation may possibly be achieved by using an ωtransaminase with poor E-value, or combining two enzymes with
opposite enantiopreference, and a catalytic amount of amino
acceptor (e.g. pyruvate). The dynamic kinetic resolution reaction
may require a two phase-system where the ω-transaminase is
dissolved in an aqueous phase and the immobilised CalB is situated
in an organic solvent phase; this would require adequate mass
transfer to function. Another approach is to attempt the complete
reaction in aqueous medium; CalB, and a variant thereof, has been
shown to perform acylation in buffer.118
48
There is also the option of testing the ω-transaminase for activity in
organic solvents, after immobilisation. In related works we
immobilised enzymes on modified silica oxide to achieve condition
promiscuity. The His6-tag was used for binding to metal ions on a
chelating surface.119,120 Initial tests with modified controlled pore
glass in place of the silica oxide were performed with ωtransaminase, activity was found in toluene, but the system remains
to be explored and optimised. Active ω-transaminase in organic
solvent will presumably display altered substrate specificity, and is
likely to provide possibilities for alternative solutions for equilibrium
displacement, although it arguably is a less environmentally
sustainable approach compared to aqueous systems.
7.2.2 Mechanistic studies
The reaction mechanism of the ω-transaminases is assumed to
follow the same pattern as proposed for aspartic α-transaminases.46
In the proposed reaction mechanism (section 1.5, Figure 6) a proton
is abstracted to form a planar quinonoid structure. When an αamino acid forms a Schiff-base with PLP the pKa is lowered for the
proton to be abstracted for formation of the quinonoid intermediate,
the presumed rate determining step. When the substrate is an
aliphatic amine this effect is less significant, the conjugated base is
stabilised by resonance in the pyridoxal moiety but not by a
carboxylate. Preliminary quantum mechanical modelling predicted
the pKa to be 14 units higher for an isopropylamine-PLP complex
compared to the alanine-PLP complex when modelled in Geobacillus
stearothermophilus alanine racemase.121 It is still unclear as to why
the ω-transaminase is able to abstract this proton, notwithstanding
having a relatively low turnover number. The charge distribution in
the active site of the ω-transaminases may lower the barrier in
question; this effect is possibly enhanced in Cv-ωTA Trp60Cys.
Crystallization experiments with 1-deazapyridoxal-5'-phosphate to
form a stabile external aldimine with chosen substrates, as
demonstrated by Toney et al. for an aspartate transaminase,79 may
be a suitable extension of this study for mapping the structure and
substrate interactions of the wild type and variant Trp60Cys. A
comparative study with a homologous amino acid racemase may
suggest feasible mutation points for creating an amino racemase
with a suitable PLP-dependent enzyme as template.
49
8 Acknowledgements
To my supervisor and mentor, Prof. Per Berglund, thank you for half
a decade (at least) of great teaching! The same goes for Prof. Karl
Hult, my enzyme guru, who never turned down an opportunity for
discussion. I will miss stumbling into your offices with questions out
of the blue.
To all my colleagues; office mates and co-workers, students and
teachers, co-authors and collaborators, I thank you for help and
friendship inside and outside of the lab. I don not think I have seen
the last of you...
To my family and extended family, thank you for all your help and
support! You made it possible to work at those so important odd
hours before a deadline. To all my friends (not least my band mates),
thanks for much needed distractions from science.
To my life companion Julia and my daughter Nadia, thank you for
encouragement, understanding and being more important to me
than anything else in the world!
Financial support from The Swedish Governmental Agency for
Innovation Systems (VINNOVA) is gratefully acknowledged. EUCOST Action CM0701 is acknowledged for travel funds and for the
opportunity to join the COST Training School in Cascade
Chemoenzymatic Processes – New Synergies between Chemistry and
Biochemistry - in Italy 2009. KTH is acknowledged for travelling
scholarships in 2006, 2007 and 2008 that enabled conference
participation.
50
9 References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. P. Grunwald, Biocatalysis: Biochemical Fundamentals and Applications, Imperial College Press, 2009. C. Burel, Industrial or White Biotechnology ‐ Research for Europe, EuropaBio, ESAB. D. Carrez and W. Soetaert, Industrial or White Biotechnology ‐ A driver of sustainable growth in Europe, EuropaBio, ESAB. D. J. Mack, M. Brichacek, A. Plichta and J. T. Njarðarson, http://cbc.arizona.edu/njardarson/group/sites/default/files/Top20
0PharmaceuticalProductsByWorldwideSalesin2009.pdf, 2009. D. Voet, J. G. Voet and C. W. Pratt, Fundamentals of Biochemistry: Life at the Molecular Level, 2nd edn., Wiley, 2006. T. Golson and B. Fink, The Farmstead Egg Cookbook, St. Martin's Press, 2006. R. Chang, Physical Chemistry for the Chemical and Biological Sciences, 3rd edn., University Science Books, 2000. T. C. Bruice, Chem Rev, 2006, 106, 3119‐3139. A. Warshel, P. K. Sharma, M. Kato, Y. Xiang, H. Liu and M. H. Olsson, Chem Rev, 2006, 106, 3210‐3235. G. G. Hammes, S. J. Benkovic and S. Hammes‐Schiffer, Biochemistry, 2011, 50, 10422‐10430. M. Nic, J. Jirat and B. Kosata, International Union of Pure and Applied Chemistry, 2011, http://goldbook.iupac.org/PDF/goldbook.pdf. H. Bisswanger, Enzyme Kinetics: Principles and Methods, Wiley‐
VCH, 2002. R. A. Copeland, Enzymes: A Practical Introduction to Structure, Mechanism and Data Analysis, 2nd edn., Wiley‐VCH, 2000. M. Jones, Organic Chemistry, 2nd edn., W. W. Norton & Company, Inc., New York, 2000. V. Henri, 1903, Hermann, Paris. L. Michaelis and M. L. Menten, Biochem Z, 1913, 49, 333‐369. L. Michaelis, M. L. Menten, K. A. Johnson and R. S. Goody, Biochemistry, 2011, 50, 8264‐8269. G. P. Moss, http://www.chem.qmul.ac.uk/iubmb/kinetics/index.html, 1981, Symbolism and Terminology in Enzyme Kinetics, Nomenclature Committee of the International Union of Biochemistry (NC‐IUB). 51
19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. G. E. Briggs and J. B. Haldane, Biochem J, 1925, 19, 338‐339. G. G. Hammes and J. L. Haslam, Biochemistry, 1969, 8, 1591‐1598. C. S. Chen, Y. Fujimoto, G. Girdaukas and C. J. Sih, J Am Chem Soc, 1982, 104, 7294‐7299. C. S. Chen, S. H. Wu, G. Girdaukas and C. J. Sih, J Am Chem Soc, 1987, 109, 2812‐2817. N. Ran, E. Rui, J. Liu and J. Tao, Curr Pharm Des, 2009, 15, 134‐152. Y. S. Ding and J. S. Fowler, Drug Dev Res, 2003, 59, 227‐239. C. Khosla and P. B. Harbury, Nature, 2001, 409, 247‐252. F. H. Arnold, Nature, 2001, 409, 253‐257. A. Schmid, J. S. Dordick, B. Hauer, A. Kiener, M. Wubbolts and B. Witholt, Nature, 2001, 409, 258‐268. S. Lutz, Curr Opin Biotechnol, 21, 734‐743. W. P. C. Stemmer, Proc Natl Acad Sci U S A, 1994, 91, 10747‐10751. A. Koltermann and U. Kettling, Biophys Chem, 1997, 66, 159‐177. F. H. Arnold, Acc Chem Res, 1998, 31, 125‐131. N. J. Turner, Trends Biotechnol, 2003, 21, 474‐478. F. H. Arnold and A. A. Volkov, Curr Opin Chem Biol, 1999, 3, 54‐59. M. T. Reetz, L. W. Wang and M. Bocola, Angew Chem Int Ed Engl, 2006, 45, 1236‐1241. M. T. Reetz, M. Bocola, J. D. Carballeira, D. Zha and A. Vogel, Angew Chem Int Ed Engl, 2005, 44, 4192‐4196. R. J. Kazlauskas and U. T. Bornscheuer, Nat Chem Biol, 2009, 5, 526‐529. M. S. Humble and P. Berglund, Eur J Org Chem, 2011, 3391‐3401. K. Hult and P. Berglund, Trends Biotechnol, 2007, 25, 231‐238. U. T. Bornscheuer and R. J. Kazlauskas, Angew Chem Int Ed Engl, 2004, 43, 6032‐6040. Q. Wu, B.‐K. Liu and X.‐F. Lin, Curr Org Chem, 2010, 14, 1966‐1988. N. Tokuriki and D. S. Tawfik, Science, 2009, 324, 203‐207. C. Branneby, Doctoral Thesis, Royal Institute of Technology, 2005. M. D. Toney, Arch Biochem Biophys, 2005, 433, 279‐287. M. D. Toney, Biochim Biophys Acta, 2011, 1814, 1407‐1418. E. F. Oliveira, N. M. Cerqueira, P. A. Fernandes and M. J. Ramos, J Am Chem Soc, 2011, 133, 15496‐15505. R. B. Silverman, The Organic Chemistry of Enzyme‐Catalysed Reactions, Academic Press, Elsevier Science, 2000. W. M. Patrick, J. Weisner and J. M. Blackburn, ChemBioChem, 2002, 3, 789‐792. 52
48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. J. S. Shin and B. G. Kim, Biotechnol Bioeng, 1999, 65, 206‐211. J. S. Shin and B. G. Kim, J Org Chem, 2002, 67, 2848‐2853. J. Ward and R. Wohlgemuth, Curr Org Chem, 2010, 14, 1914‐1927. B. M. Nestl, B. A. Nebel and B. Hauer, Curr Opin Chem Biol, 2011, 15, 187‐193. T. C. Nugent and M. El‐Shazly, Adv Synth Catal, 2010, 352, 753‐819. M. Höhne and U. T. Bornscheuer, ChemCatChem, 2009, 1, 42‐51. D. Koszelewski, D. Pressnitz, D. Clay and W. Kroutil, Org Lett, 2009, 11, 4810‐4812. D. Koszelewski, I. Lavandera, D. Clay, G. M. Guebitz, D. Rozzell and W. Kroutil, Angew Chem Int Ed Engl, 2008, 47, 9337‐9340. D. Koszelewski, M. Göritzer, D. Clay, B. Seisser and W. Kroutil, ChemCatChem, 2010, 2, 73‐77. D. Koszelewski, K. Tauber, K. Faber and W. Kroutil, Trends Biotechnol, 2010, 28, 324‐332. K. O. Lidström, PharmaChem, 2007, 6, 5‐7. J. D. Stewart, Curr Opin Chem Biol, 2001, 5, 120‐129. C. K. Savile, J. M. Janey, E. C. Mundorff, J. C. Moore, S. Tam, W. R. Jarvis, J. C. Colbeck, A. Krebber, F. J. Fleitz, J. Brands, P. N. Devine, G. W. Huisman and G. J. Hughes, Science, 2010, 329, 305‐309. J. S. Shin and B. G. Kim, Biotechnol Bioeng, 1998, 60, 534‐540. J. S. Shin and B. G. Kim, Biosci Biotechnol Biochem, 2001, 65, 1782‐
1788. J. S. Shin, B. G. Kim, A. Liese and C. Wandrey, Biotechnol Bioeng, 2001, 73, 179‐187. H. Yun, B. Y. Hwang, J. H. Lee and B. G. Kim, Appl Environ Microbiol, 2005, 71, 4220‐4224. H. Yun, B. K. Cho and B. G. Kim, Biotechnol Bioeng, 2004, 87, 772‐
778. M. D. Truppo, N. J. Turner and J. D. Rozzell, Chem Commun (Camb), 2009, 2127‐2129. P. Tufvesson, J. Lima‐Ramos, J. S. Jensen, N. Al‐Haque, W. Neto and J. M. Woodley, Biotechnol Bioeng, 2011, 108, 1479‐1493. B. Y. Hwang and B. G. Kim, Enzyme Microb Technol, 2004, 34, 429‐
436. A. R. Martin, R. DiSanto, I. Plotnikov, S. Kamat, D. Shonnard and S. Pannuri, Biochem Eng J, 2007, 37, 246‐255. M. Höhne, S. Kuhl, K. Robins and U. T. Bornscheuer, ChemBioChem, 2008, 9, 363‐365. 53
71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. M. D. Truppo, J. D. Rozzell, J. C. Moore and N. J. Turner, Org Biomol Chem, 2009, 7, 395‐398. D. Koszelewski, I. Lavandera, D. Clay, D. Rozzell and W. Kroutil, Adv Synth Catal, 2008, 350, 2761‐2766. P. P. Taylor, D. P. Pantaleone, R. F. Senkpeil and I. G. Fotheringham, Trends Biotechnol, 1998, 16, 412‐418. G. Matcham, M. Bhatia, W. Lang, C. Lewis, R. Nelson, A. Wang and W. Wu, Chimia, 1999, 53, 584‐589. W. Wu, M. Bhatia, C. M. Lewis, W. Lang, A. L. Wang and G. W. Matcham, US Pat., 1999, 6 133 018. G. R. Schonbaum, B. Zerner and M. L. Bender, J Biol Chem, 1961, 236, 2930‐2935. T. S. Soper and J. M. Manning, J Biol Chem, 1981, 256, 4263‐4268. D. Peisach, D. M. Chipman, P. W. Van Ophem, J. M. Manning and D. Ringe, J Am Chem Soc, 1998, 120, 2268‐2274. W. R. Griswold, A. J. Fisher and M. D. Toney, Biochemistry, 2011, 50, 5918‐5924. U. Schell, R. Wohlgemuth and J. M. Ward, J Mol Catal B Enzym, 2009, 59, 279‐285. J. H. Seo, D. Kyung, K. Joo, J. Lee and B. G. Kim, Biotechnol Bioeng, 2011, 108, 253‐263. D. Koszelewski, D. Clay, D. Rozzell and W. Kroutil, Eur J Org Chem, 2009, 2009, 2289‐2292. B. K. Cho, H. J. Cho, S. H. Park, H. Yun and B. G. Kim, Biotechnol Bioeng, 2003, 81, 783‐789. H. Yun and B. G. Kim, Biosci Biotechnol Biochem, 2008, 72, 3030‐
3033. M. D. Truppo, J. D. Rozzell and N. J. Turner, Org Process Res Dev, 2010, 14, 234‐237. V. Leskovac, S. Trivic and D. Pericin, FEMS Yeast Res, 2002, 2, 481‐
494. CambrexNorthBrunswickInc, S. Pannuri, S. Kamat and A. R. M. Garcia, World Pat, 2006, WO 2006/063336 A063332. W. Whitman and F. R. Tabita, Biochem Biophys Res Commun, 1976, 71, 1034‐1039. S. C. Chen, H. H. Daron and J. L. Aull, Int J Biochem, 1989, 21, 1217‐
1221. S. Schätzle, M. Höhne, E. Redestad, K. Robins and U. T. Bornscheuer, Anal Chem, 2009, 81, 8244‐8248. 54
91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. A. R. Martin, D. Shonnard, S. Pannuri and S. Kamat, Appl Microbiol Biotechnol, 2007, 76, 843‐851. B. K. Cho, H. Y. Park, J. H. Seo, J. Kim, T. J. Kang, B. S. Lee and B. G. Kim, Biotechnol Bioeng, 2008, 99, 275‐284. B. Y. Hwang, S. H. Ko, H. Y. Park, J. H. Seo, B. S. Lee and B. G. Kim, J Microbiol Biotechnol, 2008, 18, 48‐54. M. Höhne, S. Schätzle, H. Jochens, K. Robins and U. T. Bornscheuer, Nat Chem Biol, 2010, 6, 807‐813. F. G. Mutti, C. S. Fuchs, D. Pressnitz, J. H. Sattler and W. Kroutil, Adv Synth Catal, 2011, 353, 3227‐3233. S. Schätzle, F. Steffen‐Munsberg, A. Thontowi, M. Höhne, K. Robins and U. T. Bornscheuer, Adv Synth Catal, 2011, 353, 2439‐2445. M. Svedendahl, C. Branneby, L. Lindberg and P. Berglund, ChemCatChem, 2010, 2, 976‐980. F. G. Mutti, J. Sattler, K. Tauber and W. Kroutil, ChemCatChem, 2011, 3, 109‐111. H.‐J. Federsel, M. Hedberg, F. R. Qvarnström, M. P. T. Sjögren and W. Tian, Acc Chem Res, 2007, 40, 1377–1384. V. J. Harding and R. M. MacLean, J Biol Chem, 1916, 25, 337‐350. C. G. Swain, J Org Chem, 1984, 49, 2005‐2010. C. G. Swain and E. C. Lupton, J Am Chem Soc, 1968, 90, 4328‐4337. C. G. Swain, S. H. Unger, N. R. Rosenquist and M. S. Swain, J Am Chem Soc, 1983, 105, 492‐502. L. P. Hammett, J Am Chem Soc, 1937, 59, 96‐103. U. Kaulmann, K. Smithies, M. E. B. Smith, H. C. Hailes and J. M. Ward, Enzyme Microb Technol, 2007, 41, 628‐637. P. K. Mehta, T. I. Hale and P. Christen, Eur J Biochem, 1993, 214, 549‐561. J. N. Jansonius, Curr Opin Struct Biol, 1998, 8, 759‐769. P. Berglund, M. Svedendahl Humble and C. Branneby, in Comprehensive Chirality, eds. H. Yamamoto, E. Carreira and N. J. Turner, Elsevier, Oxford, UK, 2012, vol. 7, in press. C. Sayer, M. N. Isupov and J. A. Littlechild, Acta Crystallogr Sect F Struct Biol Cryst Commun, 2007, 63, 117‐119. S. F. Mayer, W. Kroutil and F. Kurt, Chem Soc Rev, 2001, 30, 332‐
339. R. A. Sheldon and F. van Rantwijk, Aust J Chem, 2004, 57, 281‐289. COST European Cooperation in the field of Scientific and Technical Research, 2012, CASCAT COST ACTION CM0701 Cascade Chemo‐
55
113. 114. 115. 116. 117. 118. 119. 120. 121. Enzymatic Processes: New Synergies between Chemistry and Biochemistry, http://www.cost‐cascat.polimi.it/. O. Thum and K. M. Oxenbøll, SOFW J, 2008, 134. A. I. Denesyuk, K. A. Denessiouk, T. Korpela and M. S. Johnson, J Mol Biol, 2002, 316, 155‐172. D. Logan, Personal communication, 2011. F. van Rantwijk and R. A. Sheldon, Tetrahedron, 2004, 60, 501‐519. L. K. Thalén and J. E. Bäckvall, Beilstein J Org Chem, 2010, 6, 823‐
829. M. W. Larsen, D. F. Zielinska, M. Martinelle, A. Hidalgo, L. J. Jensen, U. T. Bornscheuer and K. Hult, ChemBioChem, 2010, 11, 796‐801. K. E. Cassimjee, M. Trummer, C. Branneby and P. Berglund, Biotechnol Bioeng, 2008, 99, 712‐716. K. E. Cassimjee, R. Kourist, D. Lindberg, M. Wittrup Larsen, N. H. Thanh, M. Widersten, U. T. Bornscheuer and P. Berglund, Biotechnol J, 2011, 6, 463‐469. L. Rhong‐Zhen and F. Himo, Personal communication, 2008. 56
Appendix
Papers I to V
57