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Cloning and partial sequencing of an operon encoding two Pseudomonas putida haloalkanoate dehalogenases of opposite stereospecificity.

Biotechnology Department, ICI Pharmaceuticals, Macclesfield, Cheshire, United Kingdom.

ABSTRACT

We have cloned fragments of DNA (up to 13 kb), from Pseudomonas putida AJ1, that code for two stereospecific haloalkanoate dehalogenases. These enzymes are highly specific for D and L substrates. The two genes, designated hadD and hadL, have been isolated and independently expressed in Escherichia coli and P. putida hosts by using broad-host-range vectors. They are closely adjacent and inducible in what appears to be an operon with an upstream open reading frame of unknown function. Nucleotide sequence determination of hadD predicts a mature, cytoplasmic protein of 300 amino acid residues (molecular weight of 33,601). This has no significant homology with the L-specific haloalkanoate dehalogenases from Pseudomonas sp. strain CBS3 (B. Schneider, R. Muller, R. Frank, and F. Lingens, J. Bacteriol. 173:1530-1535, 1991) nor with any other known DNA or protein sequences.

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