Characterization of the haloacid dehalogenase from Xanthobacter autotrophicus GJ10 and sequencing of the dhlB gene - PubMed (original) (raw)

Characterization of the haloacid dehalogenase from Xanthobacter autotrophicus GJ10 and sequencing of the dhlB gene

J van der Ploeg et al. J Bacteriol. 1991 Dec.

Abstract

The haloacid dehalogenase of the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ10 was purified from a mutant with an eightfold increase in expression of the enzyme. The mutant was obtained by selecting for enhanced resistance to monobromoacetate. The enzyme was purified through (NH4)2SO4 fractionation, DEAE-cellulose chromatography, and hydroxylapatite chromatography. The molecular mass of the protein was 28 kDa as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 36 kDa as determined with gel filtration on Superose 12 fast protein liquid chromatography. The enzyme was active with 2-halogenated carboxylic acids and converted only the L-isomer of 2-chloropropionic acid with inversion of configuration to produce D-lactate. The activity of the enzyme was not readily influenced by thiol reagents. The gene encoding the haloacid dehalogenase (dhlB) was cloned and could be allocated to a 6.5-kb EcoRI-BglII fragment. Part of this fragment was sequenced, and the dhlB open reading frame was identified by comparison with the N-terminal amino acid sequence of the protein. The gene was found to encode a protein of 27,433 Da that showed considerable homology (60.5 and 61.0% similarity) with the two other haloacid dehalogenases sequenced to date but not with the haloalkane dehalogenase from X. autotrophicus GJ10.

PubMed Disclaimer

References

    1. J Bacteriol. 1991 Jan;173(1):124-9 - PubMed
    1. J Biol Chem. 1968 Jan 25;243(2):428-34 - PubMed
    1. EMBO J. 1991 Jun;10(6):1297-302 - PubMed
    1. J Bacteriol. 1991 Feb;173(4):1530-5 - PubMed
    1. Mol Biol Evol. 1985 Nov;2(6):557-67 - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources