Epoxide hydrolases catalyze the cofactor-independent hydrolysis of reactive and toxic epoxides. They play an essential role in the detoxification of various xenobiotics in higher organisms and in the bacterial degradation of several environmental pollutants. The first x-ray structure of one of these, from Agrobacterium radiobacter AD1, has been determined by isomorphous replacement at 2.1-Å resolution. The enzyme shows a two-domain structure with the core having the ␣/␤ hydrolase-fold topology. The catalytic residues, Asp 107 and His 275 , are located in a predominantly hydrophobic environment between the two domains. A tunnel connects the back of the active-site cavity with the surface of the enzyme and provides access to the active site for the catalytic water molecule, which in the crystal structure, has been found at hydrogen bond distance to His 275 . Because of a crystallographic contact, the active site has become accessible for the Gln 134 side chain, which occupies a position mimicking a bound substrate. The structure suggests Tyr 152 /Tyr 215 as the residues involved in substrate binding, stabilization of the transition state, and possibly protonation of the epoxide oxygen.Epoxide hydrolases (EC 3.3.2.3) are a group of functionally related enzymes that catalyze the cofactor-independent hydrolysis of epoxides to their corresponding diols by the addition of a water molecule. Epoxides are very reactive electrophilic compounds frequently found as intermediates in the catabolic pathway of various xenobiotics. For instance they are the carcinogens formed by bioactivation reactions catalyzed by cytochrome P450. Therefore, conversion of epoxides to less toxic, watersoluble compounds is an essential detoxification step in living cells. Consequently, epoxide hydrolases have been found in a wide variety of organisms, including mammals, invertebrates, plants, and bacteria (1).Until now most research has been focused on mammalian epoxide hydrolases (2, 3), which, together with glutathione S-transferases, are the most important enzymes to convert toxic epoxides to more polar and easily excretable compounds (4). However, much progress has recently also been made in the characterization of bacterial epoxide hydrolases (5, 6, 7). These enzymes show a significant sequence homology with those of mammalian origin. They can be easily obtained in large amounts, and they exhibit enantioselectivity with various industrially important epoxides, which makes them promising biocatalysts for the large scale preparation of enantiopure epoxides and/or their corresponding vicinal diols (8). In particular, extensive studies have been performed on the epoxide hydrolase from Agrobacterium radiobacter AD1, a Gram-negative bacterium that is able to use the environmental pollutant epichlorohydrin as its sole carbon and energy source (5,6,8). This epoxide hydrolase is a soluble monomeric globular protein of 35 kDa with a broad substrate range. Epichlorohydrin and epibromohydrin are its best substrates, and the optimum pH range for catalysis...

The L-2-haloacid dehalogenase from the 1,2-dichloroethane degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small L-2-haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids. Its crystal structure was solved by the method of multiple isomorphous replacement with incorporation of anomalous scattering information and solvent flattening, and was refined at 1.95-Å resolution to an R factor of 21.3%. The three-dimensional structure is similar to that of the homologous L-2-haloacid dehalogenase from Pseudomonas sp. YL (1), but the X. autotrophicus enzyme has an extra dimerization domain, an active site cavity that is completely shielded from the solvent, and a different orientation of several catalytically important amino acid residues. Moreover, under the conditions used, a formate ion is bound in the active site. The position of this substrate-analogue provides valuable information on the reaction mechanism and explains the limited substrate specificity of the Xanthobacter L-2-haloacid dehalogenase.The bacterium Xanthobacter autotrophicus is capable of growing on short-chain haloalkanes as its sole source of carbon and energy (2). Its natural substrate 1,2-dichloroethane is degraded via 2-chloroethanol, chloroacetaldehyde, and chloroacetate to glycolate in four successive enzymatic reactions before it enters the organism's central metabolic routes. Brominated compounds can also be processed in this way. Two different dehalogenases are used to cleave off the halogen atoms in the first and fourth step. In the first step, a haloalkane dehalogenase catalyzes the conversion of 1,2-dichloroethane into 2-chloroethanol and chloride. The three-dimensional structure (3) and catalytic mechanism (4) of this enzyme have been elucidated by x-ray crystallography.In the fourth degradation step, a 2-haloacid dehalogenase catalyzes the conversion of chloroacetate to glycolate and chloride. Over 20 different 2-haloacid dehalogenases (EC 3.8.1.2) have been found in various bacteria (5). They have been grouped into four different classes according to their substrate specificity and stereospecific action on 2-monochloropropionic acid: two classes of enzymes that are active with only the L-or D-substrate, yielding products with inversion of configuration at the chiral C-2 carbon atom. The two other classes act on both stereo-isomers, one with inversion of configuration, the other with retention. High amino acid sequence identities are observed among the dehalogenases within any of the separate classes (6, 7), but no homology is evident between the 2-haloacid dehalogenases from different classes.The 2-haloacid dehalogenase from X. autotrophicus belongs to the class of L-specific dehalogenases that act with inversion of configuration (L-DEXs).1 The dhlB gene encoding for it was cloned and sequenced, and the enzyme (DhlB) has been purified and characterized (8). The protein consists of a single polypeptide chain of 253 amino acids and has a molecular mass of 27,431 Da. The amino ac...

The L-2-haloacid dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small L-2-haloalkanoates to their corresponding D-2-hydroxyalkanoates, with inversion of the configuration at the C 2 atom. The structure of the apoenzyme at pH 8 was refined at 1.5-Å resolution. By lowering the pH, the catalytic activity of the enzyme was considerably reduced, allowing the crystal structure determination of the complexes with L-2-monochloropropionate and monochloroacetate at 1.7 and 2.1 Å resolution, respectively. Both complexes showed unambiguous electron density extending from the nucleophile Asp 8 to the C 2 atom of the dechlorinated substrates corresponding to a covalent enzyme-ester reaction intermediate. L-2-Haloacid dehalogenase (L-DEX) 1 catalyzes the hydrolytic dehalogenation of L-2-haloalkanoates to the corresponding D-2-hydroxyalkanoates with inversion of the configuration at the C 2 atom. Several homologous L-DEXs have been found in various Pseudomonas species and in Xanthobacter autotrophicus GJ10, a bacterium that is able to degrade the xenobiotic compound 1,2-dichloroethane (1, 2). This halogenated hydrocarbon is industrially produced in large quantities and is applied as a solvent and as an intermediate in the production of plastics (3). Because microorganisms that contain dehalogenases can be used in a biotechnological approach to detoxify halogenated aliphatics (4), such enzymes are a fascinating target for research. In addition, the stereospecificity of L-DEXs could make them useful for the biosynthesis of chiral 2-hydroxyalkanoic acids. Furthermore, L-2-haloacid dehalogenase is the prototypical member of a large superfamily of hydrolases, the haloacid dehalogenase (HAD) superfamily identified by Koonin and coworkers (5, 6). Based on three conserved sequence motifs, the L-DEXs, epoxide hydrolases, P-type ATPases, and a variety of phosphatases are recognized as members of this superfamily. Detailed information on L-DEXs is of interest as the enzyme is the only member of the HAD superfamily that has been structurally characterized so far.The x-ray structures of two L-2-haloacid dehalogenases have been reported, L-DEX YL from Pseudomonas sp. YL (Protein Data Bank code 1JUD (7)) and DhlB from X. autotrophicus GJ10 (Protein Data Bank code 1AQ6 (8)). The enzymes share a sequence identity of 40%, and their structures are closely related. Both enzymes have a mixed ␣/␤ core domain in a Rossmann fold with a four-helix bundle subdomain insertion. DhlB is somewhat larger, and the 21 extra residues form a two-helix excursion from the ␣/␤ core domain on the same side as the four-helix bundle. Together these helical domains provide a tight dimer interface and limit the substrate specificity of the X. autotrophicus enzyme to short substrates such as haloacetates and halopropionates (8,9).Comprehensive biochemical data have been obtained for the Pseudomonas enzyme (1, 10, 11).2 Asp 8 was identified as the nucleophile in the first step of the ...

Kinetic characterization and X-ray structure of a mutant of haloalkane dehalogenase with higher catalytic activity and modified substrate range Schanstra, Joost P.; Ridder, Ivo S.; Heimeriks, Gaston J.; Rink, Rick; Poelarends, Gerrit; Kalk, Kor H.; Dijkstra, Bauke W.; Janssen, Dick IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document VersionPublisher's PDF, also known as Version of record Publication date : 1996 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Schanstra, J. P., Ridder, I. S., Heimeriks, G. J., Rink, R., Poelarends, G. J., Kalk, K. H., ... Janssen, D. B. (1996). Kinetic characterization and X-ray structure of a mutant of haloalkane dehalogenase with higher catalytic activity and modified substrate range. Biochemistry, 35(40), 13186-13195. DOI: 10.1021/bi961151a Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. ABSTRACT: Conversion of halogenated aliphatics by haloalkane dehalogenase proceeds via the formation of a covalent alkyl-enzyme intermediate which is subsequently hydrolyzed by water. In the wild type enzyme, the slowest step for both 1,2-dichloroethane and 1,2-dibromoethane conversion is a unimolecular enzyme isomerization preceding rapid halide dissociation. Phenylalanine 172 is located in a helixloop-helix structure that covers the active site cavity of the enzyme, interacts with the Cl of 1,2-dichloroethane during catalysis, and could be involved in stabilization of this helix-loop-helix region of the cap domain of the enzyme. To obtain more information about the role of this residue in dehalogenase function, we performed a mutational analysis of position 172 and studied the kinetics and X-ray structure of the Phe172Trp enzyme. The Phe172Trp mutant had a 10-fold higher k cat /K m for 1-chlorohexane and a 2-fold higher k cat for 1,2-dibromoethane than the wild-type enzyme. The X-ray structure of the Phe172Trp enzyme showed a local conformational change in the helix-loop-helix region that covers the active site. This could explain the elevated activity for 1-chlorohexane of the Phe172Trp enzyme, since it allows this large substrate to bind more easily in the active site cavity. Pre-steady-state kinetic analysis showed that the increase in k cat found for 1,2-dibromoethane conversion could be attributed to an increase in the...

The large HAD (haloacid dehalogenase) superfamily of hydrolases comprises P-type ATPases, phosphatases, epoxide hydrolases and L-2-haloacid dehalogenases. A comparison of the three-dimensional structure of L-2-haloacid dehalogenase with that of the response regulator protein CheY allowed the assignment of a conserved pair of aspartate residues as the Mg2+-binding site in the P-type ATPase and phosphatase members of the superfamily. From the resulting model of the active site, a conserved serine/threonine residue is suggested to be involved in phosphate binding, and a mechanism comprising a phosphoaspartate intermediate is postulated.