Nucleotide sequence of the gene encoding cis-biphenyl dihydrodiol dehydrogenase (bphB) and the expression of an active recombinant His-tagged bphB gene product from a PCB degrading bacterium, Pseudomonas putida OU83

Abstract

The nucleotide sequence of the bphB gene of Pseudomonas putida strain OU83 was determined. The bphB gene, which encodes cis-biphenyl dihydrodiol dehydrogenase (BDDH), was composed of 834 base pairs with an ATG initiation codon and a TGA termination codon. It can encode a polypeptide of 28.91 kDa, containing 277 amino acids. Promoter-like and ribosome-binding sequences were identified upstream of the bphB gene. The bphB nucleotide sequence was used to produce His-tagged BDDH, in Escherichia coli. The His-tagged BDDH construction, carrying a single 6 x His tail on the N-terminal portion, was active. The molecular mass of the native enzyme was 128 kDa and on SDS-PAGE analysis the molecular mass was 31 kDa. This enzyme requires NAD+ for its activity and its optimum pH is 8.5. Nucleotide and the deduced amino acid sequence analyses revealed a high degree of homology between the bphB gene from Pseudomonas putida OU83 and the bphB genes from P. cepacia LB400 and P. pseudoalcaligenes KF707.