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Abstract: Enantioselective epoxide hydrolases are useful biocatalysts for the preparation of enantiopure epoxides and diols. The kinetic resolution of racemic epoxides can be carried out in an organic/aqueous biphasic system to allow use of high epoxide concentrations. Enzyme inactivation in such a system, however, may occur by contact with the interface. In this study, we investigated the factors which influence the interfacial inactivation of Agrobacterium radiobacter epoxide hydrolase in an octane/water biphasic system. Rates of interfacial inactivation were measured both in a stirred-cell, which has a planar interface, and in an emulsion reactor. Interfacial inactivation rates measured in the stirred-cell at a fixed interfacial area increased with mixing intensity. Interfacial inactivation rates per unit area were lower in the emulsion reactor than in the stirred-cell and increased with bulk aqueous enzyme concentration. Circular dichroism measurements showed that during biphasic incubation all unadsorbed soluble enzyme existed in the native conformation. Activity assays showed that the dissolved enzyme was also fully active, indicating that inactivated enzyme precipitated from solution. Using an inactive epoxide hydrolase mutant structurally similar to the wild-type enzyme in order to avoid the conversion of the epoxide, it was found that high concentrations of epoxide in the organic phase increased the rate of interfacial inactivation. [Copyright &y& Elsevier]

Mechanistic insight into the biochemistry of carbon–halogen bond cleavage is rapidly growing because of recent structural, biochemical and computational studies that have provided further insight into how haloalkane dehalogenases achieve their impressive catalytic activity. An occluded water-free active-site cavity together with strong hydrogen bond donating groups reduce the transition state energy barrier compared with that of the non-enzymatic reaction in water. Even though all known haloalkane dehalogenases belong to the α/β-hydrolase fold family, there are interesting differences in mechanistic and kinetic details, as shown by properties of mutant enzymes and transient-state kinetic studies. To improve enzymatic degradation of some environmentally important recalcitrant compounds, site-directed mutagenesis and directed-evolution studies are being done. [Copyright &y& Elsevier]

Epoxides are attractive intermediates for producing chiral compounds. Important biocatalytic reactions involving epoxides include epoxide hydrolase mediated kinetic resolution, leading to the formation of diols and enantiopure remaining substrates, and enantioconvergent enzymatic hydrolysis, which gives high yields of a single enantiomer from racemic mixtures. Epoxides can also be converted by non-hydrolytic enantioselective ring opening, using alternative anionic nucleophiles; these reactions can be catalysed by haloalcohol dehalogenases. The differences in scope of these enzymatic conversions is related to their different catalytic mechanisms, which involve, respectively, covalent catalysis with an aspartate carboxylate as the nucleophile and non-covalent catalysis with a tyrosine that acts as a general acid-base. The emerging new possibilities for enantioselective biocatalytic conversion of epoxides suggests that their importance in green chemistry will grow. [Copyright &y& Elsevier]

Highlights: [•] Targeting critical sites for stability improvement. [•] Consensus/ancestral sequence based protein stabilization. [•] Computational design of thermostability. [•] Loop grafting and chimeras for stabilization. [Copyright &y& Elsevier]

Chiral β-amino acids occur as constituents of various natural and synthetic compounds with potentially useful bioactivities. The pyridoxal 5'-phosphate (PLP)-dependent S-selective transaminase from Mesorhizobium sp. strain LUK (MesAT) is a fold type I aminotransferase that can be used for the preparation of enantiopure β-Phe and derivatives thereof. Using x-ray crystallography, we solved structures of MesAT in complex with (S)-β- Phe, (R)-3-amino-5-methylhexanoic acid, 2-oxoglutarate, and the inhibitor 2-aminooxyacetic acid, which allowed us to unveil the molecular basis of the amino acid specificity and enantioselectivity of this enzyme. The binding pocket of the side chain of aβ-amino acid is located on the 3'-oxygen side of the PLP cofactor. The same binding pocket is utilized by MesAT to bind the β-carboxylate group of an β-amino acid. A β-amino acid thus binds in a reverse orientation in the active site of MesAT compared with an α-amino acid. Such a binding mode has not been reported before for any PLP-dependent aminotransferase and shows that the active site of MesAT has specifically evolved to accommodate both β- and α-amino acids. [ABSTRACT FROM AUTHOR]

A gene encoding an alditol oxidase was found in the genome of Streptomyces coelicolor A3(2). This newly identified oxidase, AldO, was expressed at extremely high levels in Escherichia coli when fused to maltose-binding protein. AldO is a soluble monomeric flavoprotein with subunits of 45.1 kDa, each containing a covalently bound FAD cofactor. From sequence alignments with other flavoprotein oxidases, it was found that AldO contains a conserved histidine (His46) that is typically involved in covalent FAD attachment. Covalent FAD binding is not observed in the H46A AldO mutant, confirming its role in covalent attachment of the flavin cofactor. Steady-state kinetic analyses revealed that wild-type AldO is active with several polyols. The alditols xylitol (Km = 0.32 mMi, κcat = 13 s-1) and sorbitol (Km = 1.4 mM, κcat = 17 s-1) are the preferred substrates. From pre-steady-state kinetic analyses, using xylitol as substrate, it can be concluded that AldO mainly follows a ternary complex kinetic mechanism. Reduction of the flavin cofactor by xylitol occurs at a relatively high rate (99 s-1), after which a second kinetic event is observed, which is proposed to represent ring closure of the formed aldehyde product, yielding the hemiacetal of D-xylose. Reduced AldO readily reacts with molecular oxygen (1.7 × 105 M-1 s-1), which confirms that the enzyme represents a true flavoprotein oxidase. [ABSTRACT FROM AUTHOR]

Abstract: Penicillin acylase (PA) from Escherichia coli can catalyze the acylation of 6-aminopenicillanic acid (6-APA), a conversion that is applicable in the biocatalytic preparation of semi-synthetic β-lactam antibiotics such as ampicillin. The efficiency of this kinetically controlled conversion, in which an amide or ester acts as the acyl donor, is dependent on the kinetic properties of the enzyme. To further improve the synthetic properties of PAs, family gene shuffling was performed with the PA-encoding genes of the PAs from E. coli, Kluyvera cryocrescens and Providencia rettgeri. Of these three PAs, the E. coli enzyme possessed the best properties for the synthesis of ampicillin. Shuffled recombinant libraries were pre-screened for activity by growth selection, followed by testing the catalytic performance in ampicillin synthesis using HPLC. Three clones with improved synthetic properties were selected and sequence analysis showed that the shuffled genes were hybrids of the PA-encoding genes from E. coli and K. cryocrescens, with additional point mutations. The hybrid enzymes displayed a 40–90% increase in the relative rate of acyl transfer to the β-lactam nucleus during ampicillin synthesis. This increase was not accompanied by a reduction of synthetic activity that has previously been reported for mutants of E. coli PA constructed by site-directed mutagenesis. Similar improvements in acyl transfer were obtained for the synthesis of amoxicillin, cephalexin and cefadroxil, making the new hybrid enzymes interesting candidates for the biocatalytic synthesis of several β-lactam antibiotics. [Copyright &y& Elsevier]

Abstract: Rhamnolipids produced by Pseudomonas aeruginosa have been proposed as soil washing agents for enhanced removal of metal and organic contaminants from soil. A potential limitation for the application of rhamnolipids is sorption by soil matrix components. The objective of this study is to empirically determine the contribution of representative soil constituents (clays, metal oxides, and organic matter) to sorption of the rhamnolipid form most efficient at metal complexation (monorhamnolipid). Sorption studies show that monorhamnolipid (R1) sorption is concentration dependent. At low R1 concentrations that are relevant for enhancing organic contaminant biodegradation, R1 sorption followed the order: hematite (Fe2O3)>kaolinite>MnO2 ≈illite≈Ca-montmorillonite>gibbsite (Al(OH)3)>humic acid-coated silica. At high R1 concentrations, relevant for use in complexation/removal of metals or organics, R1 sorption followed the order: illite>>humic acid-coated silica>Ca-montmorillonite>hematite>MnO2 >gibbsite≈kaolinite. These results allowed prediction of R1 sorption by a series of six soils. Finally, a comparison of R1 and R2 (dirhamnolipid) shows that the R1 form sorbs more strongly alone than when in a mixture of both the R1 and R2 forms. The information presented can be used to estimate, on an individual soil basis, the extent of rhamnolipid sorption. This is important for determining: (1) whether rhamnolipid addition is a feasible remediation option and (2) the amount of rhamnolipid required to efficiently remove the contaminant. [Copyright &y& Elsevier]

Abstract: A new penicillin acylase was isolated by cloning and functional screening of DNA isolated from a sand soil enrichment culture. Sequence analysis of this enzyme, PAS2, revealed homology to a group of prominent penicillin G acylases, including the intensively studied enzyme of Escherichia coli ATCC 11105. Accordingly, PAS2 was found to be an Ntn-hydrolase with an N-terminal serine as the catalytic nucleophile, located on its 61.9kDa β-subunit. The α-subunit was shown to have a molecular mass of 25.5kDa.To evaluate the biocatalytic performance of the new enzyme, the complex kinetic parameters α, β 0, and γ were determined for the kinetically controlled synthesis of a number of important semi-synthetic penicillins and cephalosporins. While α is a measure for the relative affinity of the enzyme for the activated acyl donor (AD), β 0 and γ quantify the efficiency of acyl-transfer to the β-lactam nucleophile. Compared to the penicillin acylase of E. coli, PAS2 showed superior potential for the synthesis of 6-aminopenicillanic acid (6-APA)-derived antibiotics, allowing the accumulation of up to 2.3-fold more target product at significantly higher conversion rates. In the synthesis of amoxicillin, for instance, 1.6-fold more antibiotic was formed using the new enzyme, making PAS2 an interesting candidate for biocatalytic application. [Copyright &y& Elsevier]

α-Amylase from Bacillus paralicheniformis (Bli Amy), belonging to GH13_5 subfamily of glycoside hydrolases, was proven to be a highly efficient raw starch digesting enzyme. The ability of some α-amylases to hydrolyze raw starch is related to the existence of surface binding sites (SBSs) for polysaccharides that can be distant from the active site. Crystallographic studies performed on Bli Amy in the apo form and of enzyme bound with different oligosaccharides and oligosaccharide precursors revealed binding of these ligands to one SBS with two amino acids F257 and Y358 mainly involved in complex formation. The role of this SBS in starch binding and degradation was probed by designing enzyme variants mutated in this region (F257A and Y358A). Kinetic studies with different substrates show that starch binding through the SBS is disrupted in the mutants and that F257 and Y358 contributed cumulatively to binding and hydrolysis. Mutation of both sites (F257A/Y358A) resulted in a 5-fold lower efficacy with raw starch as substrate and at least 5.5-fold weaker binding compared to the wild type Bli Amy, suggesting that the ability of Bli Amy to hydrolyze raw starch with high efficiency is related to the level of its adsorption onto starch granules. • Structure determination of Bacillus paralicheniformis α-amylase ATCC 9945a • Binding of four different oligosaccharides and oligosaccharide precursors • Confirmation of the unusual starch binding site on the α-amylase • Verification of the starch binding site by mutational analysis [ABSTRACT FROM AUTHOR]

Biocatalytic peptide synthesis will benefit from enzymes that are active at low water levels in organic solvent compositions that allow good substrate and product solubility. To explore the use of proteases from thermophiles for peptide synthesis under such conditions, putative protease genes of the subtilase class were cloned from Thermus aquaticus and Deinococcus geothermalis and expressed in Escherichia coli . The purified enzymes were highly thermostable and catalyzed efficient peptide bond synthesis at 80 °C and 60 °C in neat acetonitrile with excellent conversion (>90%). The enzymes tolerated high levels of N , N -dimethylformamide (DMF) as a cosolvent (40–50% v/v), which improved substrate solubility and gave good conversion in 5+3 peptide condensation reactions. The results suggest that proteases from thermophiles can be used for peptide synthesis under harsh reaction conditions. [ABSTRACT FROM AUTHOR]

Ammonia lyases (AL) and aminomutases (AM) are emerging in green synthetic routes to chiral amines and an AL is being explored as an enzyme therapeutic for treating phenylketonuria and cancer. Although the restricted substrate range of the wild-type enzymes limits their widespread application, the non-reliance on external cofactors and direct functionalization of an olefinic bond make ammonia lyases attractive biocatalysts for use in the synthesis of natural and non-natural amino acids, including β-amino acids. The approach of combining structure-guided enzyme engineering with efficient mutant library screening has extended the synthetic scope of these enzymes in recent years and has resolved important mechanistic issues for AMs and ALs, including those containing the MIO (4-methylideneimidazole-5-one) internal cofactor. [ABSTRACT FROM AUTHOR]

Abstract: Six different bacterial haloalkane dehalogenases were recombinantly produced in Escherichia coli, purified, and used to catalyse the conversion of prochiral short-chain dihaloalkanes and a meso dihaloalkane, yielding enantioenriched haloalcohols. A two-reaction one-enzyme process was established in which the desymmetrisation of a dihaloalkane is followed by kinetic resolution of the chiral haloalcohol that is produced in the first step. In case of 1,3-dibromo-2-methylpropane and 1,3-dibromo-2-phenylpropane, an increase of the enantiomeric excess of the respective haloalcohol was observed in time, leading to ee values of >97%, with analytical yields of 24 and 52%, respectively. The results show that haloalkane dehalogenases can be used for the production of highly enantioenriched haloalcohols and that in some cases product enantiopurity can be improved by allowing a two-step one-enzyme tandem reaction. [Copyright &y& Elsevier]

Aminomutases carry out the chemically challenging exchange of a hydrogen atom and an amine substituent present on neighboring carbon atoms. In recent years, aminomutases have been intensively investigated for their biophysical, structural and mechanistic characteristics. The reactions catalyzed by these enzymes have considerable potential for biotechnological applications. Here, we present an overview of this diverse group of enzymes, with a focus on enzymatic mechanisms and recent developments in their use in applied biocatalysis. [ABSTRACT FROM AUTHOR]

Abstract: In epoxide hydrolase from Agrobacterium radiobacter (EchA), phenylalanine 108 flanks the nucleophilic aspartate and forms part of the substrate-binding pocket. The influence of mutations at this position on the activity and enantioselectivity of the enzyme was investigated. Screening for improved enantioselectivity towards para-nitrophenyl glycidyl ether (pNPGE) using spectrophotometric progress curve analysis yielded five different mutants with 3- to 7-fold improved enantioselectivity. The increase in enantioselectivity was in most cases the result of an enhanced catalytic efficiency toward the preferred enantiomer. Several mutations at position F108 resulted in a higher activity toward cis-disubstituted meso-epoxides, which were converted to a single product enantiomer. Mutant F108C converted cis-2,3-epoxybutane to (2R,3R)-2,3-butanediol of >99% ee with a 7-fold improved activity, and mutant F108A hydrolyzed cyclohexene oxide to (1R,2R)-1,2-cyclohexanediol of >99% ee with a more than 150-fold higher activity than wild-type enzyme. It is concluded that single amino acid substitutions in the active site of epoxide hydrolase can result in enzyme variants with catalytic properties that are suitable for preparative scale production of (S)-epoxides and chiral vicinal diols in high yield and with excellent ee. [Copyright &y& Elsevier]

Putrescine oxidase from Rhodococcus erythropolis NCIMB 11540 (PuORh) is a soluble homodimeric flavoprotein of 100 kDa, which catalyzes the oxidative deamination of putrescine and some other aliphatic amines. The initial characterization of PuORh uncovered an intriguing feature: the enzyme appeared to contain only one noncovalently bound FAD cofactor per dimer. Here we show that this low FAD/protein ratio is the result of tight binding of ADP, thereby competing with FAD binding. MS analysis revealed that the enzyme is isolated as a mixture of dimers containing two molecules of FAD, two molecules ADP, or one FAD and one ADP molecule. In addition, based on a structural model of PuORh that was built using the crystal structure of human monoamine oxidase B (MAO-B), we constructed an active mutant enzyme, PuORh A394C, that contains covalently bound FAD. These findings show that the covalent FAD-protein linkage can be formed autocatalytically and hint to a new-found rationale for covalent flavinylation: covalent flavinylation may have evolved to prevent binding of ADP or related cellular compounds, which would prohibit formation of flavinylated and functional enzyme. [ABSTRACT FROM AUTHOR]

The α-amino acid ester hydrolase (AEH) from Acetobacter turbidans is a bacterial enzyme catalyzing the hydrolysis and synthesis of β-lactam antibiotics. The crystal structures of the native enzyme, both unliganded and in complex with the hydrolysis product D-phenylglycine are reported, as well as the structures of an inactive mutant (S205A) complexed with the substrate ampicillin, and an active site mutant (Y206A) with an increased tendency to catalyze antibiotic production rather than hydrolysis. The structure of the native enzyme shows an acyl binding pocket, in which D-phenylglycine binds, and an additional space that is large enough to accommodate the β-lactam moiety of an antibiotic. In the S205A mutant, ampicillin binds in this pocket in a non-productive manner, making extensive contacts with the side chain of Tyr112, which also participates in oxyanion hole formation. In the Y206A mutant, the Tyr112 side chain has moved with its hydroxyl group toward the catalytic serine. Because this changes the properties of the β-lactam binding site, this could explain the increased β-lactam transferase activity of this mutant. [ABSTRACT FROM AUTHOR]

Abstract: We have demonstrated for the first time that d-glutamine (d-Gln) and d-glutamic acid (d-Glu) can be efficiently obtained in high ee (97% and 90%, respectively) by enzymatic kinetic resolution of d,l-Gln and d,l-Glu. This was achieved by enantioselective conversion of the l-enantiomers to their N-phenylacetyl derivatives in aqueous solution, using a mutant of penicillin-G acylase (PGA) from E. coli and phenylacetic acid methylester as the acyl donor. Kinetic modeling studies suggest that the high ee values obtained are both due to a strong enantiopreference for the l-amino acid in the deacylation step of the covalent enzyme intermediate, as well as to completeness of conversion that is transiently obtained as a result of the distinct preference of the mutant PGA for phenylacetic acid methylester over the N-phenylacetyl-l-amino acid product. For the other amino acids tested (Asn, Asp, and Ser), the highest ee values that were obtained for the remaining d-enantiomer are moderate (50–80%) because of lower enantioselectivity in the enzyme deacylation step and due to less complete conversion of the l-amino acid caused by competition for the active site between the acyl donor and the N-phenylacetyl-l-amino acid that is produced. The results demonstrate that the mutated PGA has great potential for the production of optically active d-amino acids by kinetic resolution. [Copyright &y& Elsevier]

The NADPH-dependent dimeric flavoenzyme 4-hydroxyacetophenone monooxygenase (HAPMO) catalyzes Baeyer-Villiger oxidations of a wide range of ketones, thereby generating esters or lactones. In the current work, we probed HAPMO-coenzyme complexes present during the enzyme catalytic cycle with the aim to gain mechanistic insight. Moreover, we investigated the structural role of the nicotinamide coenzyme. For these studies, we used (i) wild type HAPMO, (ii) the R339A variant, which is active but has a low affinity toward NADPH, and (iii) the R440A variant, which is inactive but has a high affinity toward NADPH. Electrospray ionization mass spectrometry was used as the primary tool to directly observe noncovalent protein-coenzyme complexes in real time. These analyzes showed for the first time that the nicotinamide coenzyme remains bound to HAPMO during the entire catalytic cycle of the NADPH oxidase reaction. This may also have implications for other homologous Baeyer-Villiger monooxygenases. Together with the observations that NADP+ only weakly interacts with oxidized enzyme and that HAPMO is mainly in the reduced form during catalysis, we concluded that NADP+ interacts tightly with the reduced form of HAPMO. We also demonstrated that the association with the coenzyme is crucial for enzyme stability. The interaction with the coenzyme analog 3-aminopyridine adenine dinucleotide phosphate (AADP+) strongly enhanced the thermal stability of wild type HAPMO. This coenzyme-induced stabilization may also be important for related enzymes. [ABSTRACT FROM AUTHOR]

Abstract: Halohydrin dehalogenase obtained from Agrobacterium radiobacter AD1, has been tested for the nitrite-mediated ring opening of epoxides. This reaction mainly leads to the formation of unstable hydroxynitrite ester intermediates, which can be further hydrolyzed to the corresponding diols. This conversion proceeds with high enantioselectivity and high regioselectivity towards styrene oxide derivatives. It has been concluded that halohydrin dehalogenase can serve as an attractive alternative to epoxide hydrolases in the preparation of enantiopure epoxides by kinetic resolution. [Copyright &y& Elsevier]

The haloalcohol dehalogenase from Agrobacterium radiobacter AD1 catalyses the reversible ring closure of vicinal haloalcohols to produce epoxides and halides. In the ring opening of epoxides, nonhalide nucleophiles such as N3− are accepted. The enantioselective irreversible ring opening of an epihalohydrin by N3−, combined with racemisation caused by a reversible ring opening by a halide, resulted in an enzymatic dynamic kinetic resolution yielding optically active (S)-1-azido-3-halo-2-propanol. With epichlorohydrin as a substrate, the rate of ring opening by N3− was higher than the rate of racemisation, resulting in a mixed kinetic resolution and dynamic kinetic resolution. With epibromohydrin as the substrate, the racemisation rate was higher than the rate of ring opening, resulting in an efficient dynamic kinetic resolution. By optimising the pH of the medium and the concentrations of N3− and Br−, the product (S)-1-azido-3-bromo-2-propanol could be obtained in 84% yield and 94% ee. An (R)-enantiomer selective ring closure of this bromoalcohol, catalysed by the same enzyme, caused a simultaneously occurring kinetic resolution, yielding when the conversion progressed, an increase in enantiopurity of (S)-1-azido-3-bromo-2-propanol to >99% ee with a yield of 77%. This compound and the ring-closed product glycidyl azide can be used as chiral synthetic building blocks. [Copyright &y& Elsevier]

EtaA is a newly identified FAD-containing monooxygenase that is responsible for activation of several thioamide prodrugs in Mycobacterium tuberculosis. It was found that purified EtaA displays a remarkably low activity with the antitubercular prodrug ethionamide. Hinted by the presence of a Baeyer-Villiger monooxygenase sequence motif in the EtaA sequence, we have been able to identify a large number of novel EtaA substrates. It was discovered that the enzyme converts a wide range of ketones to the corresponding esters or lactones via a Baeyer-Villiger reaction, indicating that EtaA represents a Baeyer-Villiger monooxygenase. With the exception of aromatic ketones (phenylacetone and benzylacetone), long-chain ketones (e.g. 2-hexanone and 2-dodecanone) also are converted. EtaA is also able to catalyze enantioselective sulfoxidation of methyl-ptolylsulfide. Conversion of all of the identified substrates is relatively slow with typical k[sub cat] values of around 0.02 s[sup -1]. The best substrate identified so far is phenylacetone (K[sub m] = 61 µM, k[sub cat] = 0.017 s[sup -1]). Redox monitoring of the flavin cofactor during turnover of phenylacetone indicates that a step in the reductive half-reaction is limiting the rate of catalysis. Intriguingly, EtaA activity could be increased by one order of magnitude by adding bovine serum albumin. This reactivity and substrate acceptance-profiling study provides valuable information concerning this newly identified prodrng activator from M. tuberculosis. [ABSTRACT FROM AUTHOR]

Haloalkane dehalogenases are bacterial enzymes capable of carbon-halogen bond cleavage in halogenated compounds. To obtain insights into the mechanism of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), we studied the steady-state and presteady-state kinetics of the conversion of the substrates 1-chlorohexane, chlorocyclohexane, and bromocyclohexane. The results lead to a proposal of a minimal kinetic mechanism consisting of three main steps: (i) substrate binding, (ii) cleavage of the carbon-halogen bond with simultaneous formation of an alkyl-enzyme intermediate, and (iii) hydrolysis of the alkyl-enzyme intermediate. Release of both products, halide and alcohol, is a fast process that was not included in the reaction mechanism as a distinct step. Comparison of the kinetic mechanism of LinB with that of haloalkane dehalogenase Dh1A from Xantobacter autotrophicus GJ10 and the haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 shows that the overall mechanisms are similar. The main difference is in the rate-limiting step, which is hydrolysis of the alkylenzyme intermediate in LinB, halide release in DhlA, and liberation of an alcohol in DhaA. The occurrence of different rate-limiting steps for three enzymes that belong to the same protein family indicates that extrapolation of this important catalytic property from one enzyme to another can be misleading even for evolutionary closely related proteins. The differences in the rate-limiting step were related to: (i) number and size of the entrance tunnels, (ii) protein flexibility, and (iii) composition of the halide-stabilizing active site residues based on comparison of protein structures. [ABSTRACT FROM AUTHOR]

Describes the gene sequence of and the 1.9-Å resolution crystal structure of the α-amino acid ester hydrolases (AEH) from Xanthomonas citri. Description of the AEH family of Β-lactam antibiotic acylases; Quaternary and monomer structures; Active site structure.