Identification of the CoA-ester intermediates and genes involved in the cleavage and degradation of the steroidal C-ring by Comamonas testosteroni TA441

Abstract

Comamonas testosteroni TA441 degrades steroids aerobically via aromatization of the A-ring accompanied by B-ring cleavage, followed by D- and C-ring cleavage. We previously revealed major enzymes and intermediate compounds in A,B-ring cleavage, {beta}-oxidation cycle of the cleaved B-ring, and partial C,D-ring cleavage process. Here, we elucidated the C-ring cleavage and the {beta}-oxidation cycle that follows. ScdL1L2, a 3-ketoacid Coenzyme A (CoA) transferase which belongs to the SugarP_isomerase superfamily, was thought to cleave the C-ring of 9-oxo-1,2,3,4,5,6,10,19-octanor-13,17-secoandrost-8(14)-ene-7,17-dioic acid-CoA ester, the key intermediate compound in the degradation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (3a-H-4 [3'-propionic acid]-7a{beta}-methylhexahydro-1,5-indanedione; HIP)-CoA ester in the previous study; however, this study suggested that ScdL1L2 is the isomerase of the derivative with a hydroxyl group at C-14 which cleaves C ring. The subsequent ring-cleaved product was indicated to be converted to 4-methyl-5-oxo-octane-1,8-dioic acid-CoA ester mainly by ORF33-encoded CoA-transferase (named ScdJ), followed by dehydrogenation by ORF21 and 22-encoded acyl-CoA dehydrogenase (named ScdM1M2). Then a water molecule is added by ScdN for further degradation by {beta}-oxidation. ScdN is considered to catalyze the last reaction in C,D-ring degradation by the enzymes encoded in the steroid degradation gene cluster tesB to tesR. IMPORTANCEStudies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain materials for steroid drugs. Steroid-degrading bacteria are globally distributed, and the role of bacterial steroid degradation in the environment as well as in human is attracting attention. The overall degradation of steroidal four rings is proposed, however there are still much to be revealed to understand the complete degradation pathway. This study aims to uncover the whole steroid degradation process in C. testosteroni, which is one of the most studied representative steroid degrading bacteria and is suitable for exploring the degradation pathway because the involvement of degradation-related genes can be determined by gene disruption.