Cloning and characterization of a cryptic haloacid dehalogenase from Burkholderia cepacia MBA4.
Burkholderia cepacia MBA4 has been shown to produce a single dehalogenase batch culture. Moreover, other cryptic dehalogenases were also detected when the cells were grown in continuous culture. In this paper, we report the cloning and characterization of one of the cryptic dehalogenases in MBA4. This cryptic haloacid dehalogenase, designated Chd1, was expressed constitutively in Escherichia coli. This recombinant Chd1 had a relative molecular weight of 58,000 and existed predominantly as a dimer. The subunits had a relative molecular weight of 27,000. Chd1 exhibited isomer specificity, being active towards the L-isomer of 2-monochloropropionic acid only. The structural gene, chd1, was isolated on a 1.7-kb PstI fragment. This fragment contains a functional promoter, because expression of chd1 in E. coli is orientation independent. The nucleotide sequence of this fragment was determined and characterized. An open reading frame of 840 bp encoding a putative peptide of 280 amino acids was identified. This corresponds closely with the size of the subunit. The nucleotide sequence of chd1 did not show any homology with those of other dehalogenase genes. Comparison of the predicted amino acid sequence, however, shows significant homology, ranging from 42 to 50%, with the amino acid sequences of many other dehalogenases. Chd1 is unusual in having a long leader sequence, a property of periplasmic enzymes.
Tsang
J S
JS
Molecular Microbiology Laboratory, Department of Botany, The University of Hong Kong, Hong Kong. jshtsang@hkucc.hku.hk
Sam
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D016956:Burkholderia cepacia / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D015698:Genomic Library; D006219:Halogens / Q000378:metabolism; D006867:Hydrolases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D006219:Halogens; D011498:Protein Precursors; D011994:Recombinant Proteins; D006867:Hydrolases; C034507:2-haloacid dehalogenase