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Whereas directed evolution and rational design by structural inspection are established tools for enzyme redesign, computational methods are less mature but have the potential to predict small sets of mutants with desired properties without laboratory screening of large libraries. We have explored the use of computational enzyme redesign to change the enantioselectivity of a highly thermostable alcohol dehydrogenase from Thermus thermophilus in the asymmetric reduction of ketones. The enzyme reduces acetophenone to (S)-1-phenylethanol. To invert the enantioselectivity, we used an adapted CASCO workflow which included Rosetta for enzyme design and molecular dynamics simulations for ranking. To correct for unrealistic binding modes, we used Boltzmann weighing of binding energies computed by a linear interaction energy approach. This computationally cheap method predicted four variants with inverted enantioselectivity, each with 6–8 mutations around the substrate-binding site, causing only modest reduction (2- to 7-fold) of kcat/KM values. Laboratory testing showed that three variants indeed had inverted enantioselectivity, producing (R)-alcohols with up to 99 % enantiomeric excess. The broad substrate range allowed reduction of acetophenone derivatives with full conversion to highly enantioenriched alcohols. The results demonstrate the use of computational methods to control ketoreductase stereoselectivity in asymmetric transformations with minimal experimental screening.

Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence-related to other ATP-dependent enzymes involved in lactam hydrolysis, like 5-oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP-dependent acetone- and acetophenone carboxylases. The caprolactamase was produced in E. coli, isolated by His-tag affinity chromatography, and subjected to functional and structural studies. Activity towards caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6-aminocaproic acid (6-ACA). Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG0 ' = 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αβ)2 tetramer and revealed an ATP-binding site in CapA and a Zn-coordinating site in CapB. Mutations in the ATP-binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A and H124A) were inactive and less thermostable than the wild-type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP-dependent lactam hydrolysis. This article is protected by copyright. All rights reserved.

We constructed an enzymatic network composed of three different enzymes for the synthesis of valuable ether amines. The enzymatic reactions are interconnected to catalyze the oxidation and subsequent transamination of the substrate and to provide cofactor recycling. This allows production of the desired ether amines from the corresponding ether alcohols with inorganic ammonium as the only additional substrate. To examine conversion, individual and overall reaction equilibria were established. Using these data, it was found that the experimentally observed conversions of up to 60% observed for reactions containing 10 mM alcohol and up to 280 mM ammonia corresponded well to predicted conversions. The results indicate that efficient amination can be driven by high concentrations of ammonia and may require improving enzyme robustness for scale-up. Biotechnol. Bioeng. 2016;113: 1853-1861. © 2016 Wiley Periodicals, Inc.

Protein engineering aimed at enhancing enzyme stability is increasingly supported by computational methods for calculation of mutant folding energies and for the design of disulfide bonds. To examine the accuracy of mutant structure predictions underlying these computational methods, crystal structures of thermostable limonene epoxide hydrolase variants obtained by computational library design were determined. Four different predicted effects indeed contributed to the obtained stabilization: (i) enhanced interactions between a flexible loop close to the N-terminus and the rest of the protein; (ii) improved interactions at the dimer interface; (iii) removal of unsatisfied hydrogen bonding groups; and (iv) introduction of additional positively charged groups at the surface. The structures of an eightfold and an elevenfold mutant showed that most mutations introduced the intended stabilizing interactions, and side-chain conformations were correctly predicted for 72-88% of the point mutations. However, mutations that introduced a disulfide bond in a flexible region had a larger influence on the backbone conformation than predicted. The enzyme active sites were unaltered, in agreement with the observed preservation of catalytic activities. The structures also revealed how a c-Myc tag, which was introduced for facile detection and purification, can reduce access to the active site and thereby lower the catalytic activity. Finally, sequence analysis showed that comprehensive mutant energy calculations discovered stabilizing mutations that are not proposed by the consensus or B-FIT methods. This article is protected by copyright. All rights reserved.

Computational enzyme design holds great promise for providing new biocatalysts for synthetic chemistry. A strategy to design small mutant libraries of complementary enantioselective epoxide hydrolase variants for the production of highly enantioenriched (S,S)-diols and (R,R)-diols is developed. Key features of this strategy (CASCO, catalytic selectivity by computational design) are the design of mutations that favor binding of the substrate in a predefined orientation, the introduction of steric hindrance to prevent unwanted substrate binding modes, and ranking of designs by high-throughput molecular dynamics simulations. Using this strategy we obtained highly stereoselective mutants of limonene epoxide hydrolase after experimental screening of only 37 variants. The results indicate that computational methods can replace a substantial amount of laboratory work when developing enantioselective enzymes.

We explored the use of a computational design framework for the stabilization of the haloalkane dehalogenase LinB. Energy calculations, disulfide bond design, molecular dynamics simulations, and rational inspection of mutant structures predicted many stabilizing mutations. Screening of these in small mutant libraries led to the discovery of seventeen point mutations and one disulfide bond that enhanced thermostability. Mutations located in or contacting flexible regions of the protein had a larger stabilizing effect than mutations outside such regions. The combined introduction of twelve stabilizing mutations resulted in a LinB mutant with a 23 degrees C increase in apparent melting temperature (T-m,T-app, 72.5 degrees C) and an over 200-fold longer half-life at 60 degrees C. The most stable LinB variants also displayed increased compatibility with co-solvents, thus allowing substrate conversion and kinetic resolution at much higher concentrations than with the wild-type enzyme.

Highlights • Cyclohexanone monooxygenase was stabilized by an in silico designed disulfide bond. • Stabilizing disulfide bonds were successfully designed based on a model structure. • The half-life at 30 °C was increased 12-fold for the mutant enzyme. • The apparent melting point was increased by 6 °C for the mutant enzyme. • The most stabilizing disulfide bond spans only one residue., Enzyme stability is an important parameter in biocatalytic applications, and there is a strong need for efficient methods to generate robust enzymes. We investigated whether stabilizing disulfide bonds can be computationally designed based on a model structure. In our approach, unlike in previous disulfide engineering studies, short bonds spanning only a few residues were included. We used cyclohexanone monooxygenase (CHMO), a Baeyer–Villiger monooxygenase (BVMO) from Acinetobacter sp. NCIMB9871 as the target enzyme. This enzyme has been the prototype BVMO for many biocatalytic studies even though it is notoriously labile. After creating a small library of mutant enzymes with introduced cysteine pairs and subsequent screening for improved thermostability, three stabilizing disulfide bonds were identified. The introduced disulfide bonds are all within 12 Å of each other, suggesting this particular region is critical for unfolding. This study shows that stabilizing disulfide bonds do not have to span many residues, as the most stabilizing disulfide bond, L323C–A325C, spans only one residue while it stabilizes the enzyme, as shown by a 6 °C increase in its apparent melting temperature.

Enzymatic amino acid synthesis: Kinetic resolution and asymmetric synthesis of various valuable 3-substituted aspartic acids, which were obtained in fair to good yields with diastereomeric ratio values of up to98:2 and enantiomeric excess values of up to99 %, by using engineered methylaspartate ammonia lyases are described. These biocatalytic methodologies for the selective preparation of aspartic acid derivatives appear to be attractive alternatives for existing chemical methods.

An aminomutase, naturally catalyzing the interconversion of (S)--phenylalanine and (R)--phenylalanine, was converted into an ammonia lyase catalyzing the nonoxidative deamination of phenylalanine to cinnamic acid by a rational single-point mutation. It could be shown by crystal structures and kinetic data that the flexibility of the lid that covers the active site decides whether the enzyme acts as a lyase or a mutase. An Arg92Ser mutation destabilized the closed conformation of the lid structure and converted the mutase into a lyase that exhibited up to 44-fold increased reaction rates in the enantioselective deamination of (R)--phenylalanine. In addition, the amination rates of cinnamic acid yielding optically pure (S)-- and (R)--phenylalanine were doubled. The applicability of the mutant enzyme for kinetic resolution and asymmetric amination could be shown by biocatalysis on a preparative scale.

To improve the efficiency and to investigate the molecular determinants that direct substrate specificity of chlorocatechol 2,3-dioxygenase CbzE(GJ31) , several mutant enzymes were constructed. Loci for substitutions of amino acids were selected by sequence comparisons as well as by homology modelling of known chlorocatechol 2,3-dioxygenases (CbzE(BASF) , CbzE(SK1) and CbzE(16-6A)). Activity measurements with various catechols showed that most of the modifications influenced activity only to a minor degree. The amino acid at position 154 seems to be located at a non-important position in the enzyme with minor extension into the substrate tunnel. Similarly, the change of related amino acids such as D95E and Y223F did not influence the catalysis since both residues are far away from the catalytic centre and the substrate tunnel. Even the modification of isoleucine to threonine in position 310, located at the outer substrate tunnel, showed a significant alteration of activities. Position 196 seems to be of higher relevance since the modification of valine to alanine, i.e. the reduction of the side-chain, produced much alteration. The amino acid is located at the interface of inner to outer substrate tunnel. CbzE(V196A) showed high relative k(cat) for 3-chlorocatechol. A pronounced increase in activity for 3-chlorocatechol resulted by the change from alanine to valine and from aspartic acid to glycine laying in the outer substrate tunnel at position 211 and 212 respectively.

Haloalkane dehalogenases from five sources were heterologously expressed in Escherichia coli, isolated, and tested for their ability to achieve kinetic resolution of racemic alpha-bromoamides, which are important intermediates used in the preparation of bioactive compounds. To explore the substrate scope, fourteen alpha-bromoamides, with different C alpha- and N-substituents, were synthesized. Catalytic activity towards eight substrates was found, and for five of these compounds the conversion proceeded with a high enantioselectivity (E value >200). In all cases, the (R)-alpha-bromoamide is the preferred substrate. Conversions on a preparative scale with a catalytic amount of enzyme (enzyme: substrate ratio less 1:50 w/w) were all completed within 17-46 h and optically pure alpha-bromoamides and alpha-hydroxyamides were isolated with good yields (31-50%). Substrate docking followed by molecular dynamics simulations indicated that the high enantioselectivity results from differences in the percentage of the time in which the substrate enantiomers are bound favourably for catalysis. For the preferred (R)-substrates, the angle between the attacking aspartate oxygen atom of the enzyme, the attacked carbon atom of the substrate, and the displaced halogen atom, is more often in the optimal range (>157 degrees) for reactivity. This can explain the observed enantioselectivity of LinB dehalogenase in a kinetic resolution experiment.

Coenzyme A ligases play an important role in metabolism by catalyzing the activation of carboxylic acids. In this study we describe the synthesis of aminoacyl-coenzyme As (CoAs) catalyzed by a CoA ligase from Penicillium chrysogenum. The enzyme accepted medium-chain length fatty acids as the best substrates, but the proteinogenic amino acids L-phenylalanine and L-tyrosine, as well as the non-proteinogenic amino acids D-phenylalanine, D-tyrosine and (R)- and (S)-beta-phenylalanine were also accepted. Of these amino acids, the highest activity was found for (R)-beta-phenylalanine, forming (R)-beta-phenylalanyl-CoA. Homology modeling suggested that alanine 312 is part of the active site cavity, and mutagenesis (A312G) yielded a variant that has an enhanced catalytic efficiency with beta-phenylalanines and D-alpha-phenylalanine. (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

The multistep catalytic process using designer cells, either added as freshly prepared suspensions or as stable lyophilized powder, and click reaction can be performed in one pot. The sequence of four reactions allows the production of both enantiomers of beta-hydroxytriazoles with high enantiomeric excess.

Haloalkane dehalogenase (DhlA) hydrolyzes short-chain haloalkanes to produce the corresponding alcohols and halide ions. Release of the halide ion from the active-site cavity can proceed via a two-step and a three-step route, which both contain slow enzyme isomerization steps. Thermodynamic analysis of bromide binding and release showed that the slow unimolecular isomerization steps in the three-step bromide export route have considerably larger transition state enthalpies and entropies than those in the other route. This suggests that the three-step route involves different and perhaps larger conformational changes than the two-step export route. We propose that the three-step halide export route starts with conformational changes that result in a more open configuration of the active site from which the halide ion can readily escape. In addition, we suggest that the two-step route for halide release involves the transfer of the halide ion from the halide-binding site in the cavity to a binding site somewhere at the protein surface, where a so-called collision complex is formed in which the halide ion is only weakly bound. No large structural rearrangements an necessary for this latter process.

11 Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands2 Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute forPharmaceutical Sciences, Utrecht University, The Netherlands

The halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) catalyzes nucleophilic ring opening of epoxides with cyanide and azide. In the case of 2,2-disubstituted epoxides, this reaction proceeds with excellent enantioselectivity (E values up to > 200), which gives, by kinetic resolution, access to various enantiopure epoxides and beta-substituted tertiary alcohols (ee up to 99%). Since the enzyme has a broad substrate range and because these tertiary alcohols are difficult to prepare in other ways, HheC is an attractive biocatalyst for the production of P-cyano and P-azido tertiary alcohols.

Chitooligosaccharide oxidase (ChitO) catalyzes the oxidation of C1 hydroxyl moieties on chitooligosaccharides and in this way displays a different substrate preference as compared to other known oligosaccharide oxidases. ChitO was identified in the genome of Fusarium graminearum and a structural model revealed that one active site residue (Q268) was likely to be involved in the recognition of the N-acetyl moiety on the chitooligosaccharide substrates. The substrate specificity of wild type ChitO and the Q268R mutant were examined and confirmed that Q268 is indeed involved in N-acetyl recognition. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A gene encoding a eugenol oxidase was identified in the genome from Rhodococcus sp. strain RHA1. The bacterial FAD-containing oxidase shares 45% amino acid sequence identity with vanillyl alcohol oxidase from the fungus Penicillium simplicissimum. Eugenol oxidase could be expressed at high levels in Escherichia coli, which allowed purification of 160 mg of eugenol oxidase from 1 L of culture. Gel permeation experiments and macromolecular MS revealed that the enzyme forms homodimers. Eugenol oxidase is partly expressed in the apo form, but can be fully flavinylated by the addition of FAD. Cofactor incorporation involves the formation of a covalent protein-FAD linkage, which is formed autocatalytically. Modeling using the vanillyl alcohol oxidase structure indicates that the FAD cofactor is tethered to His390 in eugenol oxidase. The model also provides a structural explanation for the observation that eugenol oxidase is dimeric whereas vanillyl alcohol oxidase is octameric. The bacterial oxidase efficiently oxidizes eugenol into coniferyl alcohol (KM=1.0 microM, kcat=3.1 s-1). Vanillyl alcohol and 5-indanol are also readily accepted as substrates, whereas other phenolic compounds (vanillylamine, 4-ethylguaiacol) are converted with relatively poor catalytic efficiencies. The catalytic efficiencies with the identified substrates are strikingly different when compared with vanillyl alcohol oxidase. The ability to efficiently convert eugenol may facilitate biotechnological valorization of this natural aromatic compound.

An efficient dynamic kinetic resolution protocol using a single enzyme is described. Both the kinetic resolution and substrate racemization are catalyzed by halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC). The HheC-catalyzed reaction of epibromohydrin and 2-bromomethyl-2-methyloxirane with sodium cyanate afforded 5-substituted 2-oxazolidinones in high yields (97% and 87%) and high optical purity (89% and > 99% ee) in the presence of catalytic amounts of bromide ion. These compounds are valuable building blocks with diverse synthetic applications.

Bacterial dehalogenases catalyse the cleavage of carbon-halogen bonds, which is a key step in aerobic mineralization pathways of many halogenated compounds that occur as environmental pollutants. There is a broad range of dehalogenases, which can be classified in different protein superfamilies and have fundamentally different catalytic mechanisms. Identical dehalogenases have repeatedly been detected in organisms that were isolated at different geographical locations, indicating that only a restricted number of sequences are used for a certain dehalogenation reaction in organohalogen-utilizing organisms. At the same time, massive random sequencing of environmental DNA, and microbial genome sequencing projects have shown that there is a large diversity of dehalogenase sequences that is not employed by known catabolic pathways. The corresponding proteins may have novel functions and selectivities that could be valuable for biotransformations in the future. Apparently, traditional enrichment and metagenome approaches explore different segments of sequence space. This is also observed with alkane hydroxylases, a category of proteins that can be detected on basis of conserved sequence motifs and for which a large number of sequences has been found in isolated bacterial cultures and genomic databases. It is likely that ongoing genetic adaptation, with the recruitment of silent sequences into functional catabolic routes and evolution of substrate range by mutations in structural genes, will further enhance the catabolic potential of bacteria toward synthetic organohalogens and ultimately contribute to cleansing the environment of these toxic and recalcitrant chemicals.

The Baeyer-Villiger monooxygenase (BVMO), 4-hydroxyacetophenone monooxygenase (HAPMO), uses NADPH and O(2) to oxidize a variety of aromatic ketones and sulfides. The FAD-containing enzyme has a 700-fold preference for NADPH over NADH. Sequence alignment with other BVMOs, which are all known to be selective for NADPH, revealed three conserved basic residues, which could account for the observed coenzyme specificity. The corresponding residues in HAPMO (Arg339, Lys439 and Arg440) were mutated and the properties of the purified mutant enzymes were studied. For Arg440 no involvement in coenzyme recognition could be shown as mutant R440A was totally inactive. Although this mutant could still be fully reduced by NADPH, no oxygenation occurred, indicating that this residue is crucial for completing the catalytic cycle of HAPMO. Characterization of several Arg339 and Lys439 mutants revealed that these residues are indeed both involved in coenzyme recognition. Mutant R339A showed a largely decreased affinity for NADPH, as judged from kinetic analysis and binding experiments. Replacing Arg339 also resulted in a decreased catalytic efficiency with NADH. Mutant K439A displayed a 100-fold decrease in catalytic efficiency with NADPH, mainly caused by an increased K(m). However, the efficiency with NADH increased fourfold. Saturation mutagenesis at position 439 showed that the presence of an asparagine or a phenylalanine improves the catalytic efficiency with NADH by a factor of 6 to 7. All Lys439 mutants displayed a lower affinity for AADP(+), confirming a role of the lysine in recognizing the 2'-phosphate of NADPH. The results obtained could be extrapolated to the sequence-related cyclohexanone monooxygenase. Replacing Lys326 in this BVMO, which is analogous to Lys439 in HAPMO, again changed the coenzyme specificity towards NADH. These results indicate that the strict NADPH dependency of this class of monooxygenases is based upon recognition of the coenzyme by several basic residues.

Haloalcohol dehalogenases are bacterial enzymes that catalyze the cofactor-independent dehalogenation of vicinal haloalcohols such as the genotoxic environmental pollutant 1,3-dichloro-2-propanol, thereby producing an epoxide, a chloride ion and a proton. Here we present X-ray structures of the haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1, and complexes of the enzyme with an epoxide product and chloride ion, and with a bound haloalcohol substrate mimic. These structures support a catalytic mechanism in which Tyr145 of a Ser-Tyr-Arg catalytic triad deprotonates the haloalcohol hydroxyl function to generate an intramolecular nucleophile that substitutes the vicinal halogen. Haloalcohol dehalogenases are related to the widespread family of NAD(P)H-dependent short-chain dehydrogenases/reductases (SDR family), which use a similar Ser-Tyr-Lys/Arg catalytic triad to catalyze reductive or oxidative conversions of various secondary alcohols and ketones. Our results reveal the first structural details of an SDR-related enzyme that catalyzes a substitutive dehalogenation reaction rather than a redox reaction, in which a halide-binding site is found at the location of the NAD(P)H binding site. Structure-based sequence analysis reveals that the various haloalcohol dehalogenases have likely originated from at least two different NAD-binding SDR precursors.

Baeyer-Villiger monooxygenases (BVMOs) are flavoenzymes that catalyze a remarkably wide variety of oxidative reactions such as regio- and enantioselective Baeyer-Villiger oxidations and sulfoxidations. Several of these conversions are difficult to achieve using chemical approaches. Due to their selectivity and catalytic efficiency, BVMOs are highly valuable biocatalysts for the synthesis of a broad range of fine chemicals. For a long time, only one member of this class of flavin-containing biocatalysts had been cloned and overexpressed which has limited their application for synthetic processes. Recently a number of new genes that encode BVMOs have been sequenced and overexpressed. In this paper the biocatalytic properties of recently cloned BVMOs are reviewed. Furthermore, the potential for obtaining novel BVMOs from sequenced genomes will be discussed.

Penicillin acylase of Escherichia coli catalyses the hydrolysis and synthesis of beta-lactam antibiotics. To study the role of hydrophobic residues in these reactions, we have mutated three active-site phenylalanines. Mutation of alphaF146, betaF24 and betaF57 to Tyr, Trp, Ala or Leu yielded mutants that were still capable of hydrolysing the chromogenic substrate 2-nitro-5-[(phenylacetyl)amino]-benzoic acid. Mutations on positions alphaF146 and betaF24 influenced both the hydrolytic and acyl transfer activity. This caused changes in the transferase/hydrolase ratios, ranging from a 40-fold decrease for alphaF146Y and alphaF146W to a threefold increase for alphaF146L and betaF24A, using 6-aminopenicillanic acid as the nucleophile. Further analysis of the betaF24A mutant showed that it had specificity constants (kcat/Km) for p-hydroxyphenylglycine methyl ester and phenylglycine methyl ester that were similar to the wild-type values, whereas the specificity constants for p-hydroxyphenylglycine amide and phenylglycine amide had decreased 10-fold, due to a decreased kcat value. A low amidase activity was also observed for the semisynthetic penicillins amoxicillin and ampicillin and the cephalosporins cefadroxil and cephalexin, for which the kcat values were fivefold to 10-fold lower than the wild-type values. The reduced specificity for the product and the high initial transferase/hydrolase ratio of betaF24A resulted in high yields in acyl transfer reactions.

The breakdown of dissolved TCE in a two-step bioremediation system is described. In the first reactor, the organism Methylosinus trichosporium OB3b is grown; in the second reactor, consisting of three 17-L column reactors in series, the cells degrade TCE. A special design allowed both for the addition of air (uG,s = 0.01-0. 04 mm s-1) in the conversion reactor to prevent oxygen limitation while minimizing stripping of TCE, and for the use of methane as exogenous electron donor. In two-step systems presented thus far, only formate was used (excess, 20 mM). We found formate additions could be reduced by 75% (15 degrees C), whereas small amounts of methane (0.02-0.04 mol CH4/g cells) could replace formate and led to equally optimal results. Example calculations show that up to 90% reduction in operating cost of chemicals can be obtained by using methane instead of formate. A model was developed to describe each of the conditions studied: excess formate and optimal methane addition, suboptimal formate addition and suboptimal methane addition. Using parameters obtained from independent batch experiments, the model gives a very good description of the overall TCE conversion in the two-step system. The system presented is flexible (oxygen/methane addition) and can easily be scaled up for field application. The model provides a tool for the design of an effective and low-cost treatment system based on methane addition in the conversion reactor.

Haloalkane dehalogenase catalyzes the hydrolytic cleavage of carbon-halogen bonds in short-chain haloalkanes. Two tryptophan residues of the enzyme (Trp125 and Trp175) form a halide-binding site in the active-site cavity, and were proposed to play a role in catalysis. The function of these residues was studied by replacing Trp125 with phenylalanine, glutamine or arginine and Trp175 by glutamine using site-directed mutagenesis. All mutants except Trp125-->Phe showed a more than 10-fold reduced k(cat) and much higher K-m values with 1,2-dichloroethane and 1,2-dibromoethane than the wild-type enzyme. Fluorescence quenching experiments showed a decrease in the affinity of the mutant enzymes for halide ions. The H-2 kinetic isotope effect observed with the wild-type enzyme in deuterium oxide was lost in the active mutants, except the Trp125-->Phe enzyme. The results indicate that both tryptophans are involved in stabilizing the transition state during the nucleophilic substitution reaction that causes carbon-halogen bond cleavage.

Haloalkane dehalogenase hydrolyses various 1-halon-alkanes to the corresponding alcohols by covalent catalysis with formation of an alkyl-enzyme intermediate. The carboxylate function of the nucleophilic aspartate (Asp-124) that displaces the halogen during formation of the intermediate was changed to an amide by site-directed mutagenesis (Asp-124-->Asn). Activity measurements and analysis of peptides containing the nucleophilic residue showed that the mutant enzyme was inactive, but that the activity increased by rapid deamidation of the asparagine residue, yielding wild type enzyme. There was no indication for isoaspartate formation during this process. The results suggest that a water molecule that is located close to the carboxyl function of Asp-124 in the X-ray structure is highly reactive and is responsible for the observed deamidation.

A review, with 160 references, discussing progress in catalytic asymmetric synthesis of β-amino acids since 2002.

The recombinant epoxide hydrolase from Agrobacterium radiobacter AD1 was used to obtain enantiomerically pure epoxides by means of a kinetic resolution. Epoxides such as styrene oxide and various derivatives thereof and phenyl glycidyl ether were obtained in high enantiomeric excess and in reasonable yield. The enantioselectivity (E-value) of the resolution was calculated from progress curves for styrene oxide (E=16.2) and para-chlorostyrene oxide (E=32.2).