Cloning and Sequence Analysis of a Plasmid-encoded 2-Haloacid Dehalogenase Gene from<i>Pseudomonas putida</i>No. 109

Kawasaki, Haruhiko; Toyama, Tohru; Maeda, Takuya; Nishino, Humiko; Tonomura, Kenzo

doi:10.1271/bbb.58.160

Cited by 41 publications

(25 citation statements)

References 8 publications

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“…In particular, eight successive residues (YGNPKYLI) in the region from 115 to 122 of DL-DEX are identical to those of D-2-haloacid dehalogenase. In contrast, DL-DEX showed no sequence similarity with L-2-haloacid dehalogenases (7,8,16,17,20,23).…”

Section: Resultsmentioning

confidence: 87%

“…Although the two families of dehalogenases act on opposite enantiomers of 2-haloacids, they are similar to each other in that they act exclusively on one particular enantiomer. Thus, DL-DEX differs markedly from them in this respect, although all of them dehalogenate 2-haloacids with inversion of the C 2 configuration of the substrates.Amino acid sequences of L-2-haloacid dehalogenases from various bacterial strains (7,8,16,17,20,23) as well as that of D-2-haloacid dehalogenase of P. putida AJ1 (1) have been determined. Sequences of L-2-haloacid dehalogenases are highly similar to one another (36 to 70% identity) but completely different from that of D-2-haloacid dehalogenase (17).…”

mentioning

confidence: 99%

“…Amino acid sequences of L-2-haloacid dehalogenases from various bacterial strains (7,8,16,17,20,23) as well as that of D-2-haloacid dehalogenase of P. putida AJ1 (1) have been determined. Sequences of L-2-haloacid dehalogenases are highly similar to one another (36 to 70% identity) but completely different from that of D-2-haloacid dehalogenase (17).…”

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confidence: 99%

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Bacterial DL-2-haloacid dehalogenase from Pseudomonas sp. strain 113: gene cloning and structural comparison with D- and L-2-haloacid dehalogenases

et al. 1997

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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 C 2 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.DL-2-Haloacid dehalogenase from Pseudomonas sp. strain 113 (DL-DEX) catalyzes the hydrolytic dehalogenation of both enantiomers of 2-haloalkanoic acids to produce the corresponding 2-hydroxyalkanoic acids with inversion of the C 2 configuration (14, 15). DL-DEX is a unique enzyme; it acts on the chiral carbon of the substrate enantiomers, which are used as equivalent substrates. Most other enzymes acting on the chiral carbon of substrates act on a sole enantiomer. Racemases and DL-DEX are the exceptions, although racemases differ markedly from DL-DEX in that they catalyze stereochemical but not chemical conversion of substrates. Racemases are believed to possess a single and common binding site for both substrate enantiomers. Thus, it will be interesting to clarify whether DL-DEX has a common site or distinct ones for each enantiomer in order to compare it with racemases. Different kinds of 2-haloacid dehalogenases showing different stereospecificities have been isolated from several bacterial strains (2, 6, 21). L-2-Haloacid dehalogenases specifically act on L-enantiomers of 2-haloacids to produce the corresponding D-2-hydroxy acids (11, 13, 23). D-2-Haloacid dehalogenase from Pseudomonas putida AJ1 dehalogenates D-enantiomers to form L-2-hydroxy acids (22). Although the two families of dehalogenases act on opposite enantiomers of 2-haloacids, they are similar to each other in that they act exclusively on one particular enantiomer. Thus, DL-DEX differs markedly from them in this respect, although all of them deha...

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Section: Resultsmentioning

confidence: 87%

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confidence: 99%

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Bacterial DL-2-haloacid dehalogenase from Pseudomonas sp. strain 113: gene cloning and structural comparison with D- and L-2-haloacid dehalogenases

et al. 1997

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“…A degree symbol indicates that the gene is described as silent. 23,47, and 73% for the activated sludge, the primary enrichment, and the secondary enrichment, respectively. Even with the low coverage of the activated sludge population, gram-positive 16S rRNA gene sequences accounted for a significant fraction of the sequences analyzed.…”

Section: Discussionmentioning

confidence: 99%

“…These sequences formed their own clade, which was more closely related to the sequence encoding the dehalogenase from P. putida No. 109 (23) than to the sequence encoding any other dehalogenase. Within this clade the level of sequence identity was Ͼ97% between dehII DP1 and dehII DP2 ; dehII DP3 was 80 and 82% identical to dehII DP2 and dehII DP1 , respectively.…”

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confidence: 99%

Comparing the Dehalogenase Gene Pool in Cultivated α-Halocarboxylic Acid-Degrading Bacteria with the Environmental Metagene Pool

Marchesi

Weightman

2003

Appl Environ Microbiol

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Culture-dependent and culture-independent approaches were used to determine the relationship between the dehalogenase gene pool in bacteria enriched and isolated on 2,2-dichloropropionic acid (22DCPA) and the environmental metagene pool (the collective gene pool of both the culturable and uncultured microbes) from which they were isolated. The dehalogenases in the pure-cultures isolates, which were able to degrade 22DCPA, were similar to previously described group I and II dehalogenases. Significantly, the majority of the dehalogenases isolated from activated sludge by degenerate PCR with primers specific for ␣-halocarboxylic acid dehalogenases were not closely related to the dehalogenases in any isolate. Furthermore, the dehalogenases found in the pure cultures predominated in the enrichments but were a minor component of the community used to inoculate the batch cultures. Phylogenetic analysis of the dehalogenase sequences isolated by degenerate PCR showed that the diversity of the group II deh gene was greater than that of the group I deh gene. Direct plating of the activated sludge onto minimal media supplemented with 22DCPA resulted in biomass and DNA from which dehalogenases were amplified. Analysis of the sequences revealed that they were much more closely related to the sequences found in the community used to start the enrichments. However, no pure cultures were obtained with this isolation method, and thus no pure cultures were available for identification. In this study we examined the link between genes found in pure cultures with the metagene pool from which they were isolated. The results show that there is a large bias introduced by culturing, not just in the bacteria isolated but also the degradative genes that they contain. Moreover, our findings serve as a caveat for studies involving the culturing of pure cultures of bacteria and conclusions which are drawn from analysis of these organisms.

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Halocompounds

Robinson¹,

Jackman²,

Stratford³

2000

Biotechnology

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Cloning and Sequence Analysis of a Plasmid-encoded 2-Haloacid Dehalogenase Gene fromPseudomonas putidaNo. 109

Cited by 41 publications

References 8 publications

Bacterial DL-2-haloacid dehalogenase from Pseudomonas sp. strain 113: gene cloning and structural comparison with D- and L-2-haloacid dehalogenases

Bacterial DL-2-haloacid dehalogenase from Pseudomonas sp. strain 113: gene cloning and structural comparison with D- and L-2-haloacid dehalogenases

Comparing the Dehalogenase Gene Pool in Cultivated α-Halocarboxylic Acid-Degrading Bacteria with the Environmental Metagene Pool

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