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.
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.
Khan
A A
AA
Microbiology Division, U.S. Food and Drug Administration, Jefferson, AR 72079, USA.
Wang
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RF
Nawaz
M S
MS
Cerniglia
C E
CE
D000595:Amino Acid Sequence; D001483:Base Sequence; D006639:Histidine; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics*; D016958:Pseudomonas putida / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis
D011994:Recombinant Proteins; D006639:Histidine; D010088:Oxidoreductases; C048338:trans-1,2-dihydrobenzene-1,2-diol dehydrogenase