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 xylosox idans ssp. dentirificans ABIV encoding a D,L-2-haloalkanoic acid halid ohydrolase (DhlIV). A 6.5-kb EcoRI/SalI-fragment with inducible expres sion 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-fragm ent 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 indi cated similarity between dhlIV and the D,L isomer specific dehI dehalo genase gene from Pseudomonas putida PP3. There are some indications fr om restriction enzyme patterns and initial sequencing data, that a gen e encoding a sigma(54)-dependent activator protein, similar to the deh R(I) regulatory gene from Pseudomonas putida PP3 is located upstream o f dhlIV. In contrast to DehI, dehalogenation of D- or L-chloropropioni c acid by the DhlIV-protein leads to lactic acid of inverted configura tion.