Crystal Structure of L-2-Haloacid Dehalogenase from Pseudomonas sp. YL

Abstract

L-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of L-2-haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids. The crystal structure of the homodimeric enzyme from Pseudomonas sp. YL has been determined by a multiple isomorphous replacement method and refined at 2.5 A resolution to a crystallographic R-factor of 19.5%. The subunit consists of two structurally distinct domains: the core domain and the subdomain. The core domain has an alpha/beta structure formed by a six-stranded parallel beta-sheet flanked by five alpha-helices. The subdomain inserted into the core domain has a four helix bundle structure providing the greater part of the interface for dimer formation. There is an active site cavity between the domains. An experimentally identified nucleophilic residue, Asp-10, is located on a loop following the amino-terminal beta-strand in the core domain, and other functional residues, Thr-14, Arg-41, Ser-118, Lys-151, Tyr-157, Ser-175, Asn-177, and Asp-180, detected by a site-directed mutagenesis experiment, are arranged around the nucleophile in the active site. Although the enzyme is an alpha/beta-type hydrolase, it does not belong to the alpha/beta hydrolase fold family, from the viewpoint of the topological feature and the position of the nucleophile.

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“…High-resolution crystal structures of HAD superfamily members [haloacid dehalogenases from Pseudomonas sp. YL and Xanthobacter autotrophicus GJ10 (30,31), the Ca 2ϩ -ATPase from rabbit sarcoplasmic reticulum (32), and PSP from Methanococcus jannaschii (33)] indicate that the catalytic domains of these proteins form a typical ␣͞␤ Rossmann fold that is similar to that in CheY (29). The quintet of conserved residues noted above surrounds the active site, as it does in receiver domains.…”

Insights into the Mechanism of Catalysis by the P−C Bond-Cleaving Enzyme Phosphonoacetaldehyde Hydrolase Derived from Gene Sequence Analysis and Mutagenesis

“…strain YL. As shown by the X-ray crystal structure of the apoenzyme represented in Figure 3, this dehalogenase folds into a small, four-R-helix bundle domain and a larger core domain containing a six-strand -sheet flanked by five R-helices (46). The active site is located at the domaindomain interface, which in the absence of bound substrate forms a large crevice.…”

“…In the second partial reaction catalyzed by the dehalogenase, an activated water molecule attacks the acyl carbon, forming a tetrahedral oxyanion intermediate which then eliminates the 2-hydroxyalkanoic acid product and regenerates the Asp10 carboxylate group. The results obtained from mutagenesis (48) and substrate docking experiments (46) have led investigators to suggest Lys151 of motif II and Ser175, Asn177, and Asp180 of motif III (see Figure 4) as additional catalytic groups. Ser176, which is also shown in the active site picture of Figure 4, is not required for catalytic activity in the dehalogenase (the Ser176Ala mutant retains 92% of the wildtype activity), but we show it here for the purpose of further discussion provided below.…”

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

“…YL (Hisano et al 1996) and DhlB from Xanthobacter autotrophicus GJ10 (Ridder et al 1997). Both of these enzymes are homodimers with each subunit having a core domain of a Rossmann fold like six-stranded parallel -sheet flanked by five -helices and a four-helix bundle sub-domain.…”