Cloning and nucleotide sequence of a D,L-haloalkanoic acid dehalogenase encoding gene from Alcaligenes xylosoxidans ssp denitrificans ABIV

We have cloned DNA fragments of plasmid pFL40 from Alcaligenes xylosoxidans ssp. dentirificans ABIV encoding a D,L-2-haloalkanoic acid halidohydrolase (DhlIV). A 6.5-kb EcoRI/SalI-fragment with inducible expression of the halidohydrolase was cloned in Pseudomonas fluorescens and Escherichia coli. A 1.9-kb HindII-fragment demonstrated expression of the dehalogenase only due to the presence of the promoter from the pUC vector in Escherichia coli. The nucleotide sequence of this DNA-fragment was determined. It had an open reading frame coding for 296 amino acid residues (molecular weight of 32783 D). The dhlIV a short segment of a D-specific dehalogenase (hadD) from Pseudomonas putida AJ1, but not to any other known DNA sequences. Restriction enzyme patterns indicated similarity between dhlIV and the D,L isomer specific dehI dehalogenase gene from Pseudomonas putida PP3. There are some indications from restriction enzyme patterns and initial sequencing data, that a gene encoding a sigma(54)-dependent activator protein, similar to the dehR(I) regulatory gene from Pseudomonas putida PP3 is located upstream of dhlIV. In contrast to DehI, dehalogenation of D- or L-chloropropionic acid by the DhlIV-protein leads to lactic acid of inverted configuration.