ABSTRACT
Actinobacterial MaoC family enoyl coenzyme A (CoA) hydratases catalyze the addition of water across the double bond of CoA esters during steroid side chain catabolism. We determined that heteromeric MaoC type hydratases, exemplified by ChsH1-ChsH2Mtb of Mycobacterium tuberculosis and CasM-CasORjost from Rhodococcus jostii RHA1, are specific toward a 3-carbon side chain steroid metabolite, consistent with their roles in the last ß-oxidation cycle of steroid side chain degradation. Hydratases containing two fused MaoC domains are responsible for the degradation of longer steroid side chains. These hydratases, encoded in the cholesterol degradation gene clusters of M. tuberculosis and R. jostii RHA1, have broad specificity and were able to catalyze the hydration of the 5-carbon side chain of both cholesterol and bile acid metabolites. Surprisingly, the homologous hydratases from the bile acid degradation pathway have low catalytic efficiencies or no activity toward the 5-carbon side chain bile acid metabolites, cholyl-enoyl-CoA, lithocholyl-enoyl-CoA, and chenodeoxycholyl-enoyl-CoA. Instead, these hydratases preferred a cholate metabolite with oxidized steroid rings and a planar ring structure. Together, the results suggest that ring oxidation occurs prior to side chain degradation in the actinobacterial bile acid degradation pathway. IMPORTANCE Characterization of the substrate specificity of hydratases described here will facilitate the development of specific inhibitors that may be useful as novel therapeutics against M. tuberculosis and to metabolically engineer bacteria to produce steroid pharmaceuticals with desired steroid rings and side chain structures.
