Comparative studies of genes encoding thermostable L-2-halo acid dehalogenase from Pseudomonas sp. strain YL, other dehalogenases, and two related hypothetical proteins from Escherichia coli

Abstract

We have determined the nucleotide sequence of the gene encoding thermostable L-2-halo acid dehalogenase (L-DEX) from the 2-chloroacrylate-utilizable bacterium Pseudomonas sp. strain YL. The open reading frame consists of 696 nucleotides corresponding to 232 amino acid residues. The protein molecular weight was estimated to be 26,179, which was in good agreement with the subunit molecular weight of the enzyme. The gene was efficiently expressed in the recombinant Escherichia coli cells: the amount of L-DEX corresponds to about 49% of the total soluble proteins. The predicted amino acid sequence showed a high level of similarity to those of L-DEXs from other bacterial strains and haloacetate dehalogenase H-2 from Moraxella sp. strain B (38 to 57% identity) but a very low level of similarity to those of haloacetate dehalogenase H-1 from Morarella sp. strain B (10%) and haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (12%). By searching the protein amino acid sequence database, we found two E. coli hypothetical proteins similar to the Pseudomonas sp. strain YL L-DEX (21 to 22%). Various halogenated organic compounds have been synthesized and are now used as pesticides, herbicides, solvents, and so on in a large scale. Several halogen compounds such as polychlorinated biphenyls (PCBs), 1,1,1-trichloro-2-bis(chlorophenyl)ethane (DDT), and 1,1,1-trichloroethane have accumulated in the environment and cause environmental pollution. Various enzymes involved in the degradation of halogenated compounds have been found, purified, and characterized. They are categorized into reductive, oxygenative, and hydrolytic dehalogenases (15, 31, 32). 2-Halo acid dehalogenases (DEXs) catalyze the hydrolytic dehalogenation of 2-halo acids and are further classified into four groups based on their stereospecificities. L-DEX acts on L-2-halo acids to give D-2-hydroxy acids (16, 18). D-DEX catalyzes the conversion of D-2-halo acids into L-2-hydroxy acids (27). There are two different types of DL-DEXs, which act on both Land D-2-halo acids: one catalyzes the dehalogenation of Land D-2-halo acids with retention of the C2-configuration of the substrates, and the other catalyzes

Investigation of Two Evolutionarily Unrelated Halocarboxylic Acid Dehalogenase Gene Families

“…The pioneering studies of Jensen (23) and Goldman and colleagues (12) resulted in the identification and characterization of hydrolytic dehalogenases associated with the catabolism of ␣HAs. In the last decade, some 18 dehalogenase (deh) genes have been cloned and sequenced (24,28,30,40,41,52,64). However, despite the availability of molecular data, even recent attempts to classify dehalogenases have tended to focus on arbitrary characteristics such as substrate specificity, especially with optically active substrates such as 2-monochloropro-pionic acid (2MCPA) (16,29,55).…”

Diversity of α‐halocarboxylic acid dehalogenases in bacteria isolated from a pristine soil after enrichment and selection on the herbicide 2,2‐dichloropropionic acid (Dalapon)

“…Tree topologies were similar whether determined from amino acid sequence alignments of the longer or shorter partial dehI gene sequences (results not shown). The four dehI genes clustered into two distinct groupings, with high supporting bootstrap values, within the β‐subdivision of the group I deh family (Hill et al ., 1999), which contains well‐characterized dehalogenases such as dl ‐dex (Nardi‐Dei et al ., 1994). The dehI DA1 showed 89% sequence identity to dl ‐dex, whereas the dehI DA2 , dehI DA3 and dehI DA4 were identical to each other and showed less sequence identity (65%) to dl ‐dex.…”

“…°, cryptic deh gene. For the dehII tree, the accession numbers and/or references for published αHA genes used are: dhlS5I° (Köhler et al ., 1998; not deposited in EMBL); dhlVII , (unpublished); hadL , (Barth et al ., 1992); dehII PP3 , (Hill et al ., 1999); deh109 , (Kawasaki et al ., 1994); dehCI and dehCII , and respectively (Seemuller et al ., 1994); dehH2 , (Kawasaki et al ., 1992); l ‐dex , (Nardi‐Dei et al ., 1994); hdlIVa , (Murdiyatmo et al ., 1992), dhlB , (van der Ploeg et al ., 1995) and dehII DA3 , (Hill et al ., 1999).…”

Horizontal transfer of dehalogenase genes on IncP1β plasmids during bacterial adaptation to degrade α-halocarboxylic acids

“…Group II deh genes cloned and sequenced from strains X12 and X13 showed c. 60% nucleotide sequence identity to L-DEX 2-haloacid dehalogenase [40]. Phylogenetic analysis (Fig.…”