Identification of "missing links" in C- and D-ring cleavage of steroids by Comamonas testosteroni TA441

Comamonas testosteroni TA441 degrades steroids aerobically via aromatization and cleavage of the A- and B-ring, followed by D- and C-ring cleavage via b-oxidation. We previously characterized most of the above degradation steps; however, a few intermediate compounds remained unaccounted for. We hypothesized that cleavage of the D-ring at C13-17 required the ScdY hydratase and was followed by cleavage of the C-ring via the ScdL1L2 transferase. The reaction was expected to produce 6-methyl-3,7-dioxo-decane-1,10-dioic acid-Coenzyme A (CoA) ester. To verify this hypothesis, we constructed a plasmid that enabled targeted gene induction in TA441 mutant strains. The major product of ScdL1L2 was found to be 3-hydroxy-6-methyl-7-oxo-decane-1,10-dioic acid-CoA ester; whereas the substrate of ScdY was revealed to be a geminal diol, 17-dihydroxy-9-oxo-1,2,3,4,5,6,10,19-octanorandrost-8(14)-en-7-oic acid-CoA ester. This finding suggests that ScdY catalyzes the addition of a water molecule at C14 of 17-dihydroxy-9-oxo-1,2,3,4,5,6,10,19-octanorandrost-8(14)-en-7-oic acid-CoA ester, leading to D-ring cleavage at C13-17. The C9 ketone of the D-ring cleavage product is then converted to a hydroxyl group, followed by C-ring cleavage to produce 3-hydroxy-6-methyl-7-oxo-decane-1,10-dioic acid-CoA ester. Precise bacterial bile acid degradation pathway will be one of the key to investigate the gut-microbiota-brain axis, which affects human health and disease. IMPORTANCEStudies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain substrates for steroid drugs. The role of steroid-degrading bacteria in relation to human health is attracting growing attention. Comamonas testosteroni TA441 is the leading bacterial model of aerobic steroid degradation and the overall pathway has been outlined previously. However, a few intermediate compounds in C- and D-ring cleavage processes have remained unknown. Here, we identified the missing compounds and can now propose the complete A-, B-, C-, and D-ring cleavage pathway employed by steroid-degrading bacteria. This finding will facilitate the application of such microorganisms for the synthesis of specific steroid derivatives.