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University of Groningen
Mutants and homologs of cephalosporin acylase
Sio, Charles Frederik
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Chapter 2: Improved β-lactam
acylases and their use as industrial
biocatalysts
Charles F. Sio & Wim J. Quax
Current Opinion in Biotechnology 2004, 15:349-355
Chapter 2
Summary
Whereas the β-lactam acylases are traditionally used for the hydrolytic processing of
penicillin G and cephalosporin C, new and mutated acylases can be used for the
hydrolysis of alternative fermentation products as well as for the synthesis of semisynthetic β-lactam antibiotics. 3D structure determination and site-directed
mutagenesis studies have increased the understanding of the catalytic mechanism of
these enzymes. The yield of hydrolysis and synthesis has been greatly improved by
process design including immobilization of the enzyme and the use of alternative
reaction media. Significant advances have been made in the resolution of racemic
mixtures by means of stereoselective acylation / hydrolysis using β-lactam acylases.
Introduction
In nature each enzyme exhibits a remarkable specificity for the conversion of a particular
set of substrates. The introduction of these enzymes as biocatalysts in industrial processes
has resulted in new innovative processes for the production of fine chemicals in the recent
years. Driven by both environmental and economic considerations, particular progress has
been made in the area of semi-synthesis of penicillin and cephalosporin antibiotics. Not
only the replacement of the acyl side chain but also modifications of the other substituents
of the β-lactam nucleus are being performed with specific enzymes today.
The enzymes of the β-lactam acylase class are traditionally used for the deacylation of the
β-lactam compounds penicillin G and glutaryl-7-aminocephalosporanic acid (glutaryl-7ACA) to 6-aminocephalosporanic acid (6-APA) and 7-ACA. These β-lactam nuclei are
the building blocks used in the production of semi-synthetic β-lactam antibiotics
(see[11,15]). The most well known β-lactam acylase is the penicillin G acylase (PGA)
from Escherichia coli. This enzyme hydrolyses the amide bond between the primary
amino group of the β-lactam nucleus and the carboxyl group of the phenylacetic acid side
chain of penicillin G. Likewise, cephalosporin acylases hydrolyze the amide bond
between the nucleus and the aliphatic side chain of cephalosporins (Figure 1). The
elucidation of the 3D structure of E. coli PGA has revealed a unique catalytic mechanism
involving an N-terminal nucleophilic serine that is activated by the free N-terminal amino
group [57].
Although advances in the fermentation processes and the hydrolytic processes have been
made (see the reviews by Arroyo et al [87] and Elander [11]), the most interesting
manuscripts in the last few years have focused on protein engineering and directed
evolution of β-lactam acylases. This review discusses new and mutated acylases for
synthesis and hydrolysis of β-lactams and other chiral compounds.
Novel β-lactam acylases
A newly characterized β-lactam acylase originates from Achromobacter sp. CCM 4824.
The discovery of this enzyme was a result of classical screening of 2000 isolates from soil
30
Improved β-lactam acylases
Figure 1: The hydrolysis of β-lactam fermentation products by β-lactam
acylases.
A: Penicillin G is hydrolyzed by PGA to 6-aminopenicillanic acid; B: glutaryl-7-ACA is
hydrolyzed by cephalosporin acylase to 7-ACA; C: adipyl-7-ADCA is hydrolyzed by
mutants of cephalosporin acylase to 7-ADCA; D: CPC is hydrolyzed by mutants of
cephalosporin acylase to 7-ACA.
samples and the corresponding bacterium has been characterized on the basis of the 16S
RNA sequence as being 99.6 % identical to Achromobacter xylosoxidans [88]. The
enzyme was found to hydrolyze β-lactams containing an α-amino group in the side chain,
such as ampicillin and cephalexin, with a 2-fold higher activity compared to penicillin G.
It is the only acylase described so far that is able to hydrolyze ampicillin faster than
penicillin G, which is potentially interesting for the enzymatic synthesis of ampicillin,
amoxicillin and cephalexin [89].
Furthermore, the substrate specificity of the acylase enzyme from Streptomyces
lavendulae was recently investigated in detail. In accordance with its former classification
this enzyme has a high activity on Penicillin V and almost no activity on Penicillin G.
Surprisingly the highest activity was found towards substrates with octanoyl as the acyl
side chain. These results taken together point to a highly hydrophobic active site, and it
was proposed to classify this enzyme as a Penicillin K acylase [90].
Interestingly, a new class of β-lactam synthases was discovered with the amino-acid ester
hydrolases of Xanthomonas citri and Acetobacter turbidans, which show a preference for
substrates with α-amino side chains and have biochemical features distinct from the other
β-lactam acylases [91,92]. These enzymes are especially suited for synthetic reactions as
the β-lactam amide formed is not a substrate. The identification of this new class of βlactam amidase was a further investigation of ampicillin acylases that were first described
31
Chapter 2
Figure 2: Mutagenesis alters the substrate specificity of a cephalosporin
acylase.
The preferred substrate of cephalosporin acylase is glutaryl-7-ACA and activity towards
the novel β-lactam fermentation product adipyl-7-ADCA is much lower. Mutants that
exhibit an increased catalytic efficiency towards adipyl-7-ADCA were obtained by
directed evolution of a cephalosporin acylase. Shown here are the best mutants found by
random mutagenesis [5,6] and saturation mutagenesis of selected residues [100,99]. A
greater than 15-fold increase in activity was observed for the single mutant N266M.
“Triple” stands for the multiple mutant M271V+Q291K+T374S.
30 years ago [93]. The 3D structure of this enzyme has been determined recently and will
be discussed separately.
Within the past 1.5 years the number of available bacterial genome sequences has grown
from 71 to over 155 (www.ncbi.nlm.nih.gov/genomes/static/eub_g.html; April 2004).
Database mining for novel β-acylases has therefore become feasible. A recent BLAST
search using the paradigm of β-lactam acylases, E. coli PGA, as a template, revealed over
50 homologous genes in the various database libraries. However, the functional analysis
of all these new putative genes is very time consuming, and it is therefore expected that
the number of well characterized novel acylases will increase steeply in the coming years.
Protein engineering of β-lactam acylases
Mutating the existing β-lactam acylases can improve the physicochemical and catalytic
characteristics. In a rational approach, non-conserved basic surface residues of Bacillus
megaterium PGA were mutated into the small, hydrophobic residue alanine. This resulted
in a two to three-fold increased half-life at 55ºC or in 40% DMF [94]. Alternatively, the
charged residues of E. coli PGA were mutated to lysines, thereby improving the
interaction with the immobilization agent glyoxyl-agarose. The inactivation of the
immobilized enzyme was decreased four to eleven-fold depending on conditions [95].
32
Improved β-lactam acylases
With regard to the synthesis of β-lactam antibiotics, it was found that mutating several
active site residues of E. coli PGA increased the fraction of the new acyl side chain that is
coupled to the β-lactam nucleus (i.e. the synthesis to hydrolysis ratio Vs/Vh), thereby
increasing the yield of ampicillin and cephalexin synthesis two to four-fold [96]. In a
different approach, the Vs/Vh ratio was improved by DNA family shuffling, using E. coli,
Kluyvera citrophila and Providencia rettgeri PGA with the P. rettgeri PGA gene as
backbone. Chimeras exhibited a 40% improved ratio compared to P. rettgeri PGA, but the
ratio was not significantly improved compared with E. coli PGA [97].
To increase the activity of cephalosporin acylase on the β-lactam fermentation product
cephalosporin C (CPC; Figure 1), Kabsch and co-workers [52] made a model of a
cephalosporin acylase with bound glutaryl-7-ACA and CPC. Several combinations of
mutations were proposed to improve the binding of the desired substrate, but experimental
results of these mutations are yet to be published. Based on a different model, Kim and
co-workers selected active site residues that clashed with CPC. Step-wise random
mutagenesis of these residues resulted in an 8-fold increase in hydrolysis, which is,
however, still too low for industrial applications [86].
Quax and co-workers [18,98] used directed evolution to alter the substrate specificities of
cephalosporin acylase towards the novel β-lactam fermentation product adipyl-7-ADCA
(Figure 1) [16]. Random mutagenesis followed by a growth selection on adipylderivatives identified three hot-spot residues in the gene, of which one, Asn266 (which
corresponds to position 68 of the β-subunit, Asnβ68), was not identified as an possible
target for mutagenesis from the 3D structures [52,86]. The catalytic efficiency of the
selected mutants towards adipyl-7-ADCA was increased two to four-fold [18,98].
Subsequently, the effect of two of the three hot-spot residues on activity was thoroughly
analyzed through the incorporation of all 20 natural amino acids at these positions,
showing several mechanisms to improve activity [99] (Figure 2). Substitutions of Asn266
with methionine afforded an 11-fold improved catalytic efficiency (Figure 2), and,
moreover, improved activity towards CPC [100].
In contrast to these results, growth selection of mutants of E. coli PGA on glutaryl-leucine
resulted in variants with an improved interaction with the leucine moiety of the selection
substrate, but no activity towards the corresponding β-lactam compound, glutaryl-7-ACA
[81].
Crystal structures of β-lactam acylases
Recently, crystal structures of several highly homologous cephalosporin acylases have
been resolved. Although the structures are all virtually identical, some controversies
remain. The structure [59] and model [52] of glutaryl-7-ACA bound enzyme are very
similar to the structure of the native enzyme, suggesting a lock-and-key mechanism for
substrate binding. However, the binding of the substrate analog bromoacetyl-7-ACA to
the non-solvent accessible residue Trpβ4 [101] and the subsequent hydrolysis of the
bound substrate analog during crystallization [102] suggest that large conformational
changes occur upon substrate binding. Moreover, the identity of the catalytic residues is
also unclear; hydrolysis is thought to be mediated via a single catalytic residue Serβ1 [51],
a Serβ1/Hisβ23 Hisβ23/Gluβ455 double dyad [52] or a Serβ1/Hisβ23/Gluβ455 catalytic
33
Chapter 2
triad [53]. The active enzyme is either a αβ heterodimer [51], or a (αβ)2 heterotetramer
[52,53].
The autocatalytic processing of the transcribed precursor into the active enzyme, in which
a spacer peptide between the α- and β-subunit is removed, has been studied via mutants
deficient in processing. It was shown that a bound water molecule plays a pivotal role in
activating the hydroxyl group of Serβ1, which is the nucleophile in the first processing
step [53-55] (Figure 3). If this water is forced out by mutating Pheβ177 to proline, the
hydroxyl group is not positioned correctly and processing is inhibited [55]. In the nondeficient enzyme, the formed intermediate is thought to be hydrolyzed by either the same
water molecule [54,55] or via a different one following a structural rearrangement of the
spacer peptide [53]. Finally, different lengths of the removed spacer peptide have been
reported for the different homologs of cephalosporin acylase, ranging from eight to eleven
residues [53]. This clearly indicates that more work is yet to be done.
The crystal structure of the α-amino acid ester hydrolase from Xanthomonas citri was also
elucidated, clearly setting this class of enzymes apart from the other β-lactam acylases. It
consists of a homotetramer with a classical Ser-His-Asp catalytic triad instead of an Nterminal nucleophile active site residue [103]. Two aspartates and a glutamate residue
close to the active site stabilize the α-amino group of the substrate, explaining the
substrate specificity of this novel class of β-lactam amidases [91,92,103].
Mechanism of the acylase reaction
In the past year a kinetic analysis of the penicillin reaction steps was published with
emphasis on not only the hydrolysis reaction but also on the synthetic reaction [58]. The
acylation step and the deacylation step have been described as separate steps in the
reaction and it was shown that the breakdown of the acyl-enzyme is much faster than its
formation implying that the rate of hydrolysis is set entirely by the acylation step. This
information is relevant with respect to the use of β-lactam acylases for the kinetically
controlled synthesis of semi-synthetic penicillins and cephalosporins, such as ampicillin
and cephalexin. For this application the enzyme should have a low affinity for the
antibiotic compared to the acyl donor to prevent the competing hydrolysis of the product
as much as possible. This knowledge has been used to design novel variants of PGA with
an improved Vs/Vh ratio (see above). It appears that the kinetics of the acylation and
deacylation step can be changed separately, but it should be stressed that the
thermodynamic equilibrium can not be changed by protein engineering of the enzyme.
The conditions for optimal yield of the antibiotic product can be influenced by varying
conditions (pH) and the concentrations of the substrates. The complexity of these
processes has been analyzed elegantly by a neural network approach [104]. To circumvent
kinetic limitations, efforts have also been directed at precipitating the antibiotic product to
drive the reaction towards one end of the equilibrium. A theoretical description of this
heterogeneous ”aqueous solution–precipitate” system was found to match quite accurately
the experimental conversion of 86 % and 76 % for ampicillin and amoxicillin,
respectively [105]. In a further refinement, the feasibility of precipitation-driven synthesis
of a zwitterionic β-lactam antibiotic (e.g. amoxicillin) has been investigated [106]. For
amoxicillin it was found that Zn2+ ions drive the precipitation of the product. In
34
Improved β-lactam acylases
Figure 3: Two proposed mechanisms for the autocatalytic processing of
cephalosporin acylase.
Cephalosporin acylase is translated as an inactive precursor. The first maturation step is
cleavage of the Gly169-Ser170 bond, releasing the N-terminus of the active site serine
Ser170 (which becomes then the first residue of the β-subunit, Serβ1). This step was
analyzed from two structures of the non-maturating Ser170Ala mutant, A [53] and B
[55]. The hydroxyl group of Ser170 is activated by a water molecule (W1 in A and B)
and performs a nucleophilic attack on the main chain carbonyl of Gly169. The
intermediate thus formed is hydrolyzed by a water molecule. However, as the two
structures contain water molecules at different positions, either W2 (activated by
His192) is thought to perform this second nucleophilic attack (A) or W1, which is the
only water near the scissile bond in structure B. Color figure at end of thesis.
experiments where ZnSO4 was added, much higher levels of amoxicillin were reached,
but an undesirable side-effect of increasing concentrations of Zn2+ ions was that β-lactam
degradation seemed to increase [106].
35
Chapter 2
Industrial applications including immobilization
Although the enzymatic production of amide bonds can be carried out with unmodified
substrates following a thermodynamically controlled strategy, the situation is very
different using aliphatic amines or β-lactam nuclei. Whereas the pK of the amino group of
the β-lactam nuclei is below pH 5, the pK of aliphatic amines is around 9.5 or even higher.
This high pK value of the amine makes it necessary to either use a high pH value to obtain
a high percentage of nonionized amine, or to use a high concentration of organic solvent
to shift the pK of the carboxylic acid. This requirement has reduced the number of
possibilities for performing a wide range of amidation reactions, including antibiotic
synthesis.
Recently, some significant improvements in the design of thermodynamically controlled
syntheses catalyzed by PGA have been reported. On the one hand, the screening of
different co-solvents showed some of them to have a similar effect on the pK of the
carboxylic acid, but a low deleterious effect on the PGA stability. A reaction in
acetonitrile containing 8% water afforded ampicillin in up to 86% yield using the more
stable PGA from Alcaligenes faecalis [107]. Also, the yield of cephalexin could be
increased significantly by using an aqueous two-phase system containing 20% (w/w)
polyethyleen glycol (PEG) 400 and 15% (w/w) magnesium sulfate, because the inhibiting
product accumulated separate from the enzyme in the PEG phase [108]. On the other hand,
immobilization of the enzyme on solid supports increases the robustness of the enzyme
towards organic media, non-physiological pH and temperature. High yields of cephalexin
were obtained using immobilized PGA, but the process needs further refinements [109].
Outlook
The latest addition to the list of applications of the β-lactam acylases is enantioselective
acylation / hydrolysis. Whereas the side chain binding site of β-lactam acylases is
restrictive, the aminic subsite accepts a large number of structures, allowing for the
resolution of a large number of compounds. Analysis of the crystal structure of E. coli
PGA identified hydrophilic and hydrophobic parts in the substrate-binding site, restricting
the binding of aromatic substrates. Models of transition-state analogs of aromatic amino
acid methyl esters showed that the ester group of the L-enantiomer can form an additional
H-bond that cannot be formed by the ester of the D-enantiomer, explaining the observed
enantioselectivity. By contrast, binding of aliphatic amino acids allowed for more degrees
of freedom; esters of both enantiomers could form the H-bond, explaining why no
enantioselectivity was observed [110].
E. coli PGA specifically acylates the R-enantiomer of phenylglycinonitrile with phenyl
acetic acid (PAA). A low solubility of the product makes the reaction virtually irreversible
and stoichiometric, leaving a high enantiopure S-phenylglycine in solution [111]. An
extensive study of the enantioselective acylation of a β-amino acid ester showed that the
source of the immobilized enzyme, pH, concentration and type of PAA and amine, and
the reagents themselves (PAA or ethyl-PAA) influenced yield and enantioselectivity of
ethyl 3-amino-5-(trimethylsilyl)-4-pentynoate to a great extent. Highly pure free amine
36
Improved β-lactam acylases
and amide was obtained using 50 g/L phenylacetic acid as free acid, 100 g/L ethyl-3amino-5-(trimethylsilyl)-4-pentynoate as the amine and 14,000 U/L PGA-450 (Roche) as
enzyme at pH 5.7, 28ºC. Moreover, the reaction could be scaled up to 70L batches, with a
half-life of the enzyme of approximately 20 runs [112]. In addition, enantioselective
hydrolysis of an ester lacking a PAA moiety by E. coli PGA was reported [113], as well
as enantioselective hydrolysis of the amide bond or ester bond of several glutaryl coupled
substrates by a cephalosporin acylase [21]. This greatly increases the substrate range for
enantioselective reactions catalyzed by β-lactam acylases.
Other new directions of research include the incorporation of immobilized PGA in a
polyacrylamide gel (allowing for the control of penicillin G hydrolysis by applying a
specific current) [114], the coupling of PGA to an HPLC column resulting in an HPLC
enzyme reactor [115], and solid-state ampicillin synthesis by E. coli PGA in a wet powder
of reagents, immobilized enzyme, salt hydrates and a little as 10% water resulting in high
space-time yields [116]. The practical use of these findings, however, will have to be
assessed in the coming years.
Conclusion
Recent years have brought new enzymes, new applications and new insights into the
biochemical features of the β-lactam acylase family. A continuation of the stream of
innovations is expected for the coming years, with high expectations for the datamining of
microbial genomes, directed evolution of the known acylases, and alternatives for the
aqueous reaction media. For the latter approach, more stable enzymes are expected to be
designed.
37
38