Three-dimensional Structure of l-2-Haloacid Dehalogenase from Xanthobacter autotrophicus GJ10 Complexed with the Substrate-analogue Formate

Abstract

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 Structure of a Cyanobacterial Sucrose-Phosphatase Reveals the Sugar Tongs That Release Free Sucrose in the Cell

“…The salt bridge to the positively charged Lys163 side chain will decrease the pK a of the carboxylate group of Asp9, thereby increasing the nucleophilicity of atom OD2. In addition, the direct coordination of the phosphate group with the Mg 2þ ion (Figure 4C), in what we describe as the catalytic position, probably polarizes the P-O bond and increases the electrophilicity of the phophorus atom, as previously proposed for other HAD enzymes (Ridder et al, 1997). The phosphate group of Suc6P is stabilized and orientated within the active site by hydrogen bonds with the side chains of Lys163 and Asp9 (atom OD1) and the main chain nitrogen atom of Gly42.…”

“…In many HAD proteins, other residues in motif III interact with a divalent metal ion, usually Mg 2þ , as do some of the residues in motif I (Morais et al, 2000;Wang et al, 2001). The structures of several members of the HAD superfamily have been determined, including proteins of the bacterial twocomponent system, a HAD, a sarcoplasmic reticulum calcium pump, a phosphoserine phosphatase, a tRNA repair enzyme, a b-phosphoglucomutase, and a deoxyribonucleotidase (Ridder et al, 1997;Welch et al, 1998;Toyoshima et al, 2000;Wang et al, 2001;Galburt et al, 2002;Lahiri et al, 2002;RinaldoMatthis et al, 2002). These proteins share a conserved a/b-domain classified as a hydrolase fold, which is similar to the Rossmann fold.…”

Caught in the Act: The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis,

“…The structure of 2-L-haloalkanoic dehalogenase was the first of the HAD family to be defined (4,25). It is comprised of an R/ -core domain and a small R-/ -cap domain.…”

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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.…”