Abstract
Steroid degradation genes of Comamonas testosteroni TA441 are encoded in at least two gene clusters: one containing the meta-cleavage enzyme gene tesB; and another consisting of ORF18, 17, tesI, H, ORF11, 12, and tesDEFG. TesH and I are, respectively, the Δ 1- and Δ 4(5α)-dehydrogenase of the 3-ketosteroid, TesD is the hydrolase for the product of meta-cleavage reaction, and TesEFG degrade one of the product of TesD. In this report, we describe the identification of the function of ORF11 ( tesA2) and 12 ( tesA1). The TesA1- and TesA2-disrupted mutant accumulated two characteristic intermediate compounds, which were identified as 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3-HSA) and its hydroxylated derivative, 3,17-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione by MS and NMR analysis. A complementation experiment using a broad-host range plasmid showed that both TesA1 and A2 are necessary for hydroxylation of 3-HSA to 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3,4-DHSA).
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