Molecular biology of the 2-haloacid halidohydrolase IVa from Pseudomonas cepacia MBA4

Abstract

The structural gene (hdl IVa) for the Pseudomonas cepacia MBA4 2-haloacid halidohydrolase IVa (Hdl IVa) was isolated on a 1.6 kb fragment of Ps. cepacia MBA4 chromosomal DNA. The recombinant halidohydrolase was expressed in Escherichia coli and Pseudomonas putida and the structural gene was subcloned on to the tac expression vector pBTac1. High-level expression from the tac promoter was seen to be temperature-dependent, a consequence of the nucleotide sequence adjacent to the fragment encoding the halidohydrolase. The nucleotide sequence of the fragment encoding the Hdl IVa was determined and analysed. Three ATG codons were identified in one of the open reading frames and the one corresponding to the start of the hdl IVa structural gene was determined by comparison of the predicted amino acid sequences with the experimentally determined N-terminal sequences of halidohydrolase IVa. The hdl IVa gene encoded a 231-amino acid-residue protein of M(r) 25,900. The sequence and predicted structural data are discussed and comparison is made with sequence data for other halidohydrolases.