Characterization and expression of the plasmid-borne bedD gene from Pseudomonas putida ML2, which codes for a NAD(+)-dependent cis-benzene dihydrodiol dehydrogenase

被引:22
作者
Fong, KPY [1 ]
Goh, CBH [1 ]
Tan, HM [1 ]
机构
[1] NATL UNIV SINGAPORE, DEPT MICROBIOL, SINGAPORE 119260, SINGAPORE
关键词
D O I
10.1128/jb.178.19.5592-5601.1996
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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.
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页码:5592 / 5601
页数:10
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