title: Bacterial DL-2-haloacid dehalogenase from Pseudomonas sp. strain 113: gene cloning and structural comparison with D- and L-2-haloacid dehalogenases.
abstract: DL-2-Haloacid dehalogenase from Pseudomonas sp. strain 113 (DL-DEX) catalyzes the hydrolytic dehalogenation of both D- and L-2-haloalkanoic acids to produce the corresponding L- and D-2-hydroxyalkanoic acids, respectively, with inversion of the C2 configuration. DL-DEX is a unique enzyme: it acts on the chiral carbon of the substrate and uses both enantiomers as equivalent substrates. We have isolated and sequenced the gene encoding DL-DEX. The open reading frame consists of 921 bp corresponding to 307 amino acid residues. No sequence similarity between DL-DEX and L-2-haloacid dehalogenases was found. However, DL-DEX had significant sequence similarity with D-2-haloacid dehalogenase from Pseudomonas putida AJ1, which specifically acts on D-2-haloalkanoic acids: 23% of the total amino acid residues of DL-DEX are conserved. We mutated each of the 26 residues with charged and polar side chains, which are conserved between DL-DEX and D-2-haloacid dehalogenase. Thr65, Glu69, and Asp194 were found to be essential for dehalogenation of not only the D- but also the L-enantiomer of 2-haloalkanoic acids. Each of the mutant enzymes, whose activities were lower than that of the wild-type enzyme, acted on both enantiomers of 2-haloacids as equivalent substrates in the same manner as the wild-type enzyme. We also found that each enantiomer of 2-chloropropionate competitively inhibits the enzymatic dehalogenation of the other. These results suggest that DL-DEX has a single and common catalytic site for both enantiomers.
authors.affiliation: Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Japan.
authors.forename: V
authors.initials: V
authors.lastname: Nardi-Dei
authors.forename: T
authors.initials: T
authors.lastname: Kurihara
authors.forename: C
authors.initials: C
authors.lastname: Park
authors.forename: N
authors.initials: N
authors.lastname: Esaki
authors.forename: K
authors.initials: K
authors.lastname: Soda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007536:Isomerism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011422:Propionates / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011817:Rabbits; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011422:Propionates; D006867:Hydrolases; C034507:2-haloacid dehalogenase