Identification of the C9-Hydrogenase for 9,17-Dioxo-1,2,3,4,10,19-Hexanorandrostan-5-oic Acid (9,17-DOHNA) and the 7α-Dehydratase Essential for Initiating β-Oxidation of the B-, C-, and D-Rings in Steroid Degradation by Comamonas testosteroni TA441

ABSTRACT Comamonas testosteroni TA441 is a model aerobic steroid-degrading bacterium whose sterane degradation pathway has been elucidated in the greatest detail to date. Similar pathways have been identified in many genera of bacteria, including both proteobacteria and actinobacteria such as Mycobacterium tuberculosis . However, the genes encoding the C9-hydrogenase for 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (9,17-DOHNA, also known as HIP) and the 7α-dehydratase essential for initiating β-oxidation of the B-, C-, and D-rings had not been identified within the gene cluster responsible for B-, C-, and D-ring degradation. In this study, we identified these missing genes, located adjacent to the chsE1E2H1H2ltp2 cluster involved in C17 side-chain degradation, and designated them scdB and scdH , respectively. This finding completes the elucidation of all degradation steps of 9,17-DOHNA prior to D-ring cleavage. AlphaFold-predicted models showed that the hydrogenases/dehydrogenases involved in steroid degradation in TA441—ScdG, ScdE, 3β-dehydrogenase (3β-DH), SteA, 3α-dehydrogenase (3α-DH), and SteB—share a similar Rossmann-like α/β/α sandwich fold with ScdB and function as dimers. In contrast, ScdH was predicted to form a homohexameric structure, similar to ScdY and ScdN, members of the crotonase-like enoyl-CoA hydratase/isomerase family involved in B-, C-, and D-ring degradation. Furthermore, AlphaFold modeling revealed that SteC, the dehydratase responsible for removing the C12β-hydroxyl group from 9,17-DOHNA derivatives, exhibits strong structural similarity to BaiE, the bile acid 7α-dehydratase of Clostridium scindens JCM 10418/VPI 12708, despite sharing only ∼28% amino acid sequence identity.