Crystal Structures of Intermediates in the Dehalogenation of Haloalkanoates by l-2-Haloacid Dehalogenase

Abstract

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 ...

Biochemical and structural studies of a l-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii

“…However the active site of the protein crystallised in the monoclinic form, which was purified in the Tris-HCl buffer, has additional density, which corresponds to a molecule with six non-hydrogen atoms. The carboxylate group of this putative compound has the same interactions as phosphate in the other crystal form and as the covalent intermediates in other known haloacid dehalogenase structures (PDB codes 1QQ7, 1QQ5; Ridder et al 1999). No additional compound with such properties was present in the purification or crystallisation buffers.…”

“…In L-DEX-YL the equivalent residue has been shown to be the acceptor of the halide (Ridder et al 1999). Studies on DhlB by Ridder et al (1997) showed that the halide stabilisation cradle was made of Arg39, Asn115 and Phe175.…”

“…In DhlB, the residues Ser114, Ala9, and Tyr10 are performing these roles (Ridder et al 1999). In S. tokodaii dehalogenase Ser95 is in the same position as the respective serine residues in L-DEX-YL and DhlB enzymes so is likely to perform this role, as are the main chains of residues Ile8 and Phe9.…”

Evolutionary Genomics of the HAD Superfamily: Understanding the Structural Adaptations and Catalytic Diversity in a Superfamily of Phosphoesterases and Allied Enzymes

“…32 A variation on this theme is seen in the haloacid dehalogenases which release a halide ion along with the formation of a regular ester linkage. 69 In the phosphonatases and sugar phosphate mutases there are differences in the initial and the terminal stages of the reaction, respectively 27,32,38,[70][71][72] (Figure 1(a)) but the core phosphoryl transfer mechanism remains the same.…”

“…Thus, the flap region was a hotspot for the insertion of the various C1 caps, which appears to suggest intense natural selection for efficient solvent exclusion. 27,32,38,69,70,73 In the case of the HAD enzymes with C2 caps there is no evidence from either biochemical or structural studies, thus far, for extensive movement of the cap itself to result in open and closed states. However, an examination of the internal cavities of the available structures of the HAD enzymes with C2 caps shows that the C2 cap forms a cavernous structure over the active site with the flap sealing off the aperture to this cavity ( Figure 7).…”