Characterization and expression of the plasmid-borne bedD gene from Pseudomonas putida ML2, which codes for a NAD+-dependent cis-benzene dihydrodiol dehydrogenase.
The catabolic plasmid pHMT112 in Pseudomonas putida ML2 contains the bed gene cluster encoding benzene dioxygenase (bedC1C2BA) and a NAD+-dependent dehydrogenase (bedD) required to convert benzene into catechol. Analysis of the nucleotide sequence upstream of the benzene dioxygenase gene cluster (bedC1C2BA) revealed a 1,098-bp open reading frame (bedD) flanked by two 42-bp direct repeats, each containing a 14-bp sequence identical to the inverted repeat of IS26. In vitro translation analysis showed bedD to code for a polypeptide of ca. 39 kDa. Both the nucleotide and the deduced amino acid sequences show significant identity to sequences of glycerol dehydrogenases from Escherichia coli, Citrobacter freundii, and Bacillus stearothermophilus. A bedD mutant of P. putida ML2 in which the gene was disrupted by a kanamycin resistance cassette was unable to utilize benzene for growth. The bedD gene product was found to complement the todD mutation in P. putida 39/D, the latter defective in the analogous cis-toluene dihydrodiol dehydrogenase. The dehydrogenase encoded by bedD) was overexpressed in Escherichia coli and purified. It was found to utilize NAD+ as an electron acceptor and exhibited higher substrate specificity for cis-benzene dihydrodiol and 1,2-propanediol compared with glycerol. Such a medium-chain dehydrogenase is the first to be reported for a Pseudomonas species, and its association with an aromatic ring-hydroxylating dioxygenase is unique among bacterial species capable of metabolizing aromatic hydrocarbons.
Fong
K P
KP
Department of Microbiology, National University of Singapore.
Goh
C B
CB
Tan
H M
HM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001482:Base Composition; D001483:Base Sequence; D005798:Genes, Bacterial; D005815:Genetic Code; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009243:NAD / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010957:Plasmids / Q000235:genetics*; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D009243:NAD; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C020054:cis-1,2-dihydrobenzene-1,2-diol dehydrogenase