Gene encoding the hydrolase for the product of the meta-cleavage reaction in testosterone degradation by Comamonas testosteroni.
In a previous study we isolated the meta-cleavage enzyme gene, tesB, that encodes an enzyme that carries out a meta-cleavage reaction in the breakdown of testosterone by Comamonas testeroni TA441 (M. Horinouchi et al., Microbiology 147:3367-3375, 2001). Here we report the isolation of a gene, tesD, that encodes a hydrolase which acts on the product of the meta-cleavage reaction. We isolated tesD by using a Tn5 mutant of TA441 that showed limited growth on testosterone. TesD exhibited ca. 40% identity in amino acid sequence with BphDs, known hydrolases of biphenyl degradation in Pseudomonas spp. The TesD-disrupted mutant showed limited growth on testosterone, and the culture shows an intense yellow color. High-pressure liquid chromatography analysis of the culture of TesD-disrupted mutant incubated with testosterone detected five major intermediate compounds, one of which, showing yellow color under neutral conditions, was considered to be the product of the meta-cleavage reaction. The methylation product was analyzed and identified as methyl-4,5-9,10-diseco-3-methoxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oate, indicating that the substrate of TesD in testosterone degradation is 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid. 4,5-9,10-Diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid was transformed by Escherichia coli-expressed TesD. Downstream of tesD, we identified tesE, F, and G, which encode for enzymes that degrade one of the products of 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid converted by TesD.
Horinouchi
Masae
M
RIKEN. JST, Wako-shi, Saitama 351-0198, Japan. masae@postman.riken.go.jp
Hayashi
Toshiaki
T
Koshino
Hiroyuki
H
Yamamoto
Takako
T
Kudo
Toshiaki
T
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D020604:Comamonas testosteroni / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D003470:Culture Media; D004251:DNA Transposable Elements; D015964:Gene Expression Regulation, Bacterial; D006867:Hydrolases / Q000235:genetics* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D013739:Testosterone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D003470:Culture Media; D004251:DNA Transposable Elements; D013739:Testosterone; C013915:boldenone; D006867:Hydrolases