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Rational Design
• Requires:
– 3D Structure of Enzyme
– Knowledge of Enzyme Mechanism
•
•
Increase size of substrate binding pocket
Add or subtract an interaction
– H-Bond
– Ion Pairing
– Disulfide Bonds
•
Replace residues with those found in homologous enzymes
• Site-Directed Mutagenesis
– Polymerase Chain Reaction
Mullis, K. et al. Science 1988, 239, 487
Site-Directed Mutagenesis
•
What to consider:
– Will the amino acid mutation change the 3D structure of the enzyme?
– Will the amino acid mutation affect the 2D structure of the enzyme
(helix breaker)?
– Is the amino acid mutation on the surface of the enzyme?
Thermolysin-like protease (TLP)
Mutazioni: A4T, T56A, G58A, T63F, S65P, A69P, G8C, and
N60C
First-order inactivation of TLPs. Stabilities of TLPs were determined at different
temperatures. Shown are the first-order inactivation curves for TLP-ste incubated at 80°C ( )
and 90°C (+), thermolysin at 90°C ( ), and the 8-fold mutant at 100°C ( )
Top and side views of the hydrophobic patches on the surfaces of
AcNIR (A) and AcPAZ (B)
Kataoka, K. et al. J. Biol. Chem. 2004;279:53374-53378
Electron transfer from Achromobacter cycloclastes
pseudoazurin (AcPAZ) to wild-type and mutant
Alcaligenes xylosoxidans nitrite reductases
(AxNIRs)
“Irrational Design”
Directed Evolution
Directed Evolution
•
Survival of the Fittest
Gene of Interest
Random Mutagenesis
etc…
Expression
etc…
Screening/Selection
Positive Mutants
DIRECTED ENZYME EVOLUTION
Error-Prone PCR
•
DNA Polymerase Fidelity
– Taq polymerase ~1 mutation every 10,000 nucleotides
•
Reduction of Fidelity
– Error rates of 0.4% to 2%
– Nearly all mutations are base substitutions
– Experimental Methods – Suboptimal Conditions
• Biased concentrations of dNTP’s
• ~ 0.5 mM – 1.0 mM Mn2+
• Increased [MgCl2] (~ 7mM)
• Addition of salt or organic solvents (DMSO)
Leung, D.W.; Chen, E.; Goeddel, D.V.; Technique 1989, 1(1), 11
Cadwell, R.C.; Joyce, G.F.; PCR Methods Appl. 1992, 2, 28
Beckman, R.A.; Mildvan, A.S.; Loeb, L.A.; Biochemistry 1985, 24, 5810
Error Bias
On average only 5.7
amino acid
substitutions are
accessible from a 1
base mutation
5’ – AUGUUACAUGGUGCACCACUUAGGCGACCAAAAUGA – 3’
5’ – AUGUUACAUGGUGCAACACUUAGGCGACCAAAAUGA – 3’
5’ – AUGUUACAUGGUGCACCUCUUAGGCGACCAAAAUGA – 3’
NH3+ - Met – Leu – His – Gly – Ala – Pro – Leu – Arg – Arg – Leu – Lys – COONH3+ - Met – Leu – His – Gly – Ala – Thr – Leu – Arg – Arg – Leu – Lys – COONH3+ - Met – Leu – His – Gly – Ala – Pro – Leu – Arg – Arg – Leu – Lys – COO-
"Asexual" Evolution by Sequential Rounds of Random Mutagenesis
Attività selezionata
Ma l’attività contro un altro
substrato varia casualmente…
Activities and thermostabilities (as measured by the ratio of residual to initial activity, in arbitrary
units) of a library of randomly mutagenized pNB esterases. Parent pNB esterases all lie within
the circle, showing that the screen is sensitive to changes brought about by primarily single
amino acid substitutions. Variants exhibiting improvements in single properties are observed
much more frequently than variants with improvements in both properties simultaneously.
Thermostabilizing mutations tend to deactivate, while activating mutations tend to destabilize.
Individual populations (in the boxes) can be recombined to obtain enzymes that are both more
active and more stable
Increased stability enhances
evolvability of the P450 BM3 heme
domain. (A) The stable 5H6 protein
yielded more mutants with new or
improved activity than the
marginally stable 21B3 protein. The
counts above the bars give the
number of improved mutants of the
total number of mutants screened.
(B) Some of the improved 5H6
mutants were greatly destabilized
relative to the parent protein,
whereas the stabilities of the
improved 21B3 mutants clustered
around those of the parent protein
(circles show T50 values for
improved mutants).
• Screening criteria
is important
• Stability can be
used instead of
improvement
• Allows for
functionally
neutral mutations
"Sexual" Evolution by Gene Recombination
Starting from a single parent sequence, random mutagenesis methods (for example,
using error-prone PCR) generate a library of genes containing point mutations.
Recombination generates gene libraries with different combinations of the mutations
from a pool of parent sequences. The parent sequences can be positive mutants from a
round of screening, genes evolved separately for different properties, or even closely
related natural sequences
Lineage of pNB esterase variants showing amino acid substitutions accumulated by four
generations of sequential random mutagenesis (fourth generation) and by DNA shuffling (fifth
and sixth generations) and screening. All variants contain amino acid substitutions H322R,
Y370F, M358V and L144M from the third generation parent
Directed Evolution: recombination protocols
DNA shuffling
Top ss of homologous
Double stranded template genes
Random
fragmentation
Recombination-dependent
Amplification PCR
Double stranded template genes
PCR
with DNaseI
PCR
-
1. denaturation 94ºC
2. Short annealing 5 s
Annealing of
3. Short extension 3s 72ºC
homologous
regions
40 cycles
Chimeras
Recombination
Recombination
PCR
Ness JE. et al. (2002) Nat Biotechnol
20:1251.
PCR- amplification
Changing the ss
templates
for 10 cycles
Ikeuchi A. et al (2003). Biotechnology
Progress. 19:1460.
Directed Evolution
•
Survival of the Fittest
Gene of Interest
Random Mutagenesis
etc…
Expression
etc…
Screening/Selection
Positive Mutants
Screening
• High-Throughput Methods
• Selection
– UV/Vis Spectroscopy
– IR Spectroscopy
– Capillary Array
Electrophoresis
– GC
– pH indicators
– Fluorescence
– Circular Dichroism
– Mass Spectrometry
Angew. Chem. Int. Ed. Engl. 1997, 36, 2830
Angew. Chem. Int. Ed. Engl. 1998, 37, 2647
Angew. Chem. Int. Ed. Engl. 2000, 39, 3891
Catal. Today 2001, 67, 389
• Phage Display
Chem. Eur. J. 1998, 4, 2324
Angew. Chem. Int. Ed. Engl. 1999, 38, 497
Angew. Chem. Int. Ed. Engl. 1999, 111, 1868
Selection
O
O
OH
O
O
O
O
Yeast Esterase
H2O
O
O
+
O
O
+
HO
O
•
Host cell experiences a growth advantage as a consequence of producing
enzyme with desired property
•
Measurement of optical density (OD) of cells identifies hits
(S)-substrate
Inhibition of Cell Growth
OD = 0.11
(R)-substrate
Cell Growth
OD = 0.51
Reetz, M.; Rüggeberg, C.J.; Chem. Commun. 2002, 1428
Phage Display
•
Phages
– Viruses that infect bacterial cells (E. coli)
– Accommodate, replicate segments of “foreign” DNA
– Foreign DNA is spliced into the gene for a phage coat protein
•
•
Affinity Selection
Enzymes Displayed
– Trypsin, Prostate Specific Antigen, β-lactamase, Lysozyme
Smith, G.P.; Petrenko, V.A.; Chem. Rev., 1997, 97, 391
O
High-Throughput Screening
OH
Hydrolyase
O
O
O
Buffer, pH 7.2
O
+
-O
O
+
H+
O
Inactive Hydrolyase
Active, non-selective
(R)-selective
(S)-selective
S ester
R ester
•
96-well plate
•
pH indicator
– p-nitrophenol (pKa = 7.15)
•
Substrates
– ~20 μg/well
– Variation between
quadruplicate measurements
< 2%
Janes, L.E.; Löwendahl, A.C. Kazlauskas, R.J.; Chem. Eur. J. 1998, 4(11), 2324
• LC-MS
High-Throughput Screening
D
•
•
•
•
•
O
D
O
O
Epoxide Hydrolase
O
Auto-sampler
+
D
D
96-well microtiter plateD
Electrospray Ionization, TOF Detector
Eight Channels
14 minutes/plate = ~10,000/day
D
OH
O
Schrader, W.; Eipper, A; Pugh, D.J.; Reetz, M.; Can. J. Chem. 2002, 80, 626
OH +
D
OH
O
D
D
D
OH
Evoluzione di una idrossilasi degli
acidi grassi (P450) in una
monoossigenasi del propano
. (a) Total turnover numbers for
propane hydroxylation (moles
propanol per mole P450). (b) Km
values and (c) catalytic rate
constants ( k cat ) for propane
oxidation. (d) ... (e) residual activity
on palmitate and laureate (f)
thermal stability (g) …
Rudi Fasan , Yergalem T. Meharenna , Christopher D. Snow , Thomas L. Poulos , Frances H. Arnold
Journal of Molecular Biology, Volume 383, Issue 5, 2008, 1069 - 1080
Fig. 2 Evolution of a P450 propane monooxygenase from a long-chain
fatty acid hydroxylase. (a) Substrate specificity profiles of P450 PMO and
its evolutionary intermediates on the C 2 –C 10 alkane series.
Fig. 5 Structure of variant 139-3. (a) Superposition of NPG-bound structures of P450 BM3 (orange, PDB code <ce:inter-ref
href="genbank:1JPZ" type="simple"> 1JPZ</ce:inter-ref> <ce:cross-ref refid="bib41"> 41 </ce:cross-ref> ) and evolutionary
intermediat...
Fig. 6 Substrate access pathway. (a) Volumetric restriction and compartmentalization of the substrate access pathway during the
transition of P450 BM3 to P450 PMO (left). The A328F mutation in 35E11 interrupts the contiguity of the path by defining an in...
Toluene oMonooxygenase
or “TOM”
• Polymer of 1500 units (amino acids)
• At each position there are 19 possibilities
(20 building blocks, 1 already used)
• 191500 possibilities (101918)
Error-Prone DNA Shuffling (Sexual PCR)
PCR w/o primers
W/T or error-prone PCR Monooxygenase
5’
3’
Anneal
3’
5’
DNase I (Random digestion)
DNA synthesis
(Pfu polymerase)
1st cycle
5’
Denature
3’
2nd cycle
5’
10-50 bp size fragments
3’
5’
5’
3’
3rd cycle 3’
5’
CentriSep column
3’
5’ Avr II
3’
30 cycles
Ppu MI 3’
5’
PCR w/ primers
5’
3’
3’
5’
3’
5’
Fragments cloned
into suitable vector
5’
Taq/pfu
polymerase
5’
3’
5’
3’
5’
3’
Avr II
Ppu MI
3’
5’
3’
5’
3’
3’
5’
5’
Resulting genes include different
mutations and combinations of mutations
Spectrophotometric Assay Methods
1. Naphthol Formation
OH
TOM
OH
Tetrazotized
o-Dianisidine
R
N
PURPLE DIAZO DYES
N
96-Well Plate Screening for Positive Variants
DNA Shuffling Created
TOM Variants
Only Colored Colonies
Were Picked
(Visual Screening)
96-well Plate Screening
Steps:
Positive variants
analyzed further
1. Growth of variants in 96well plates
2. Cells were washed and
contacted with gas phase
substrate (e.g. TCE vapor)
Spectrophotometric Detection
3. Supernatant was collected
via filtration plates and
reagents were added for color
development.
Controlling the Regiospecific Ring-Hydroxylation
of Toluene
CH3
OH
Wild-type TpMO: 90% p-cresol
o-cresol (80%)
10% m-cresol
G
07
1
A
CH3
CH3
I100S/G103S
m-cresol (75%)
Toluene
OH
A1
07
T
CH3
p-cresol (98%)
A. Fishman, Y. Tao, L. Rui, and T. K.
Wood, J. Biol. Chem. 280:506-514, 2005.
OH
Active Site Modeling: Product Docking
FeA
FeA
FeB
FeB
Wild type TpMO
TbuA1 A107G
Swiss-Pdb Viewer
ToMO Catalytic Residues
E214
I100
A101
F205
Q141
T201
E103
Fe
M180
Fe
A107
A110
Novel Compounds from Toluene Monooxygenases
Product
Enzyme
2-naphthol
T4MO I100A
nitrohydroquinone
ToMO E214G
Structure
Application
50,000 ton/yr,
no domestic source
OH
dephostatin
(Alzhiemer’s &
Parkinson’s)
NO2
OH
OH
3,6-dihydroxyfluorene &
2,6-dihydroxynaphthalene
T4MO
1-, 3-, & 4hydroxyfluourene
TOM &
TOM V106A
4-methylresorcinol*
ToMO F205G
?
HO
OH
OH
?
aromatic polyesters
$200,000/kg
OH
CH3
*Previously seen in a consortium but no enzymes identified
OH
O
H
N
O2
N
H
2H2O
O
indigo
2H2O 3/2O2
NAD+ NADH
O
2
DH
NA
oxidation
N
H
3-hydroxyindole
(indoxyl)
H
O
NA2
D+
ox
ida
tio
nb
yT
OM
dimerization
C3
Color
Chemistry
OH
O
O
dimerization
O
O2
tryptophan
N
H
2-hydroxyindole
NH
O
indirubin
H
N
O
OH
HN
N
H
2H2O
3/2O2 2H2O
NADH NAD+
try
pto
ph
an
ase
oxidation
M
TO
CH2
by
OH
O +
H2 D
NA
n
io
at
id
ox
CH C
O2 H
D
-2
C
O
H2 N
N
H
isatin
NA
N
H
indole
dimerization
O
O2
2H2O
isoindigo
N
H
Colored Compounds Extracted from TOM Variants
After Saturation Mutagenesis at Position 113
Standard:
H
N
O
H
N
O
O
O
O
N
H
O
Indigo (Blue)
N
H
N
H
NH
O
Isatin (Yellow)
Indirubin (Pink)
O
N
H
Isoindigo(Brown)
Rui, Reardon, and Wood, Appl. Microbiol. Biotechnol. 66: 422-429, 2005.
Conclusioni
•Il DNA shuffling può essere usato per individuare quali
amino acidi sono coinvolti nella catalisi
•La mutagenesi della TpMO alle posizioni I100, G103, e
A107 permette di modificare la regiochimica
dell’ossidazione di toluene e naftalene, ottenendo anche il
meta-cresolo, che gli enzimi naturali non producono
•La mutazione della A113 nella toluene monossigenasi
modifica la regiospecificità dell’ossidazione dell’indolo,
generando diversi colori
•Uso come catalizzatori per chimica fine