Multiplicity of 3-Ketosteroid-9α-Hydroxylase Enzymes in Rhodococcus rhodochrous DSM43269 for Specific Degradation of Different Classes of Steroids

Abstract

The well-known large catabolic potential of rhodococci is greatly facilitated by an impressive gene multiplicity. This study reports on the multiplicity of kshA, encoding the oxygenase component of 3-ketosteroid 9␣-hydroxylase, a key enzyme in steroid catabolism. Five kshA homologues (kshA1 to kshA5) were previously identified in Rhodococcus rhodochrous DSM43269. These KshA DSM43269 homologues are distributed over several phylogenetic groups. The involvement of these KshA homologues in the catabolism of different classes of steroids, i.e., sterols, pregnanes, androstenes, and bile acids, was investigated. Enzyme activity assays showed that all KSH enzymes with KshA DSM43269 homologues are C-9 ␣-hydroxylases acting on a wide range of 3-ketosteroids, but not on 3-hydroxysteroids. KshA5 appeared to be the most versatile enzyme, with the broadest substrate range but without a clear substrate preference. In contrast, KshA1 was found to be dedicated to cholic acid catabolism. Transcriptional analysis and functional complementation studies revealed that kshA5 supported growth on any of the different classes of steroids tested, consistent with its broad expression induction pattern. The presence of multiple kshA genes in the R. rhodochrous DSM43269 genome, each displaying unique steroid induction patterns and substrate ranges, appears to facilitate a dynamic and fine-tuned steroid catabolism, with C-9 ␣-hydroxylation occurring at different levels during microbial steroid degradation.Rhodoccoci are capable of degrading a wide range of organic compounds (15,32). This strong catabolic potential is encoded by an extremely large genome, of Ͼ9.7 Mb in the case of Rhodococcus jostii RHA1, which also carries numerous gene homologues for various enzyme classes (20). Multiple steroid catabolic gene clusters, for example, have been identified in R. jostii RHA1 (19,20,33). In particular, several homologous genes encoding key enzymes involved in steroid ring opening have been identified, i.e., 3-ketosteroid 9␣-hydroxylase (KSH), encoded by kshA and kshB, and 3-ketosteroid ⌬1-dehydrogenase (KSTD), encoded by kstD (13, 33). Hydroxylation of steroid substrates at the C-9 position, together with dehydrogenation of the A-ring performed by KSTD, leads to opening of the steroid polycyclic ring structure and the formation of 3-hydroxy-9,10-secoandrost-1,3,5(10)-triene-9,17-dione (3-HSA) (7, 31). Knowledge of steroid catabolic enzymes is limited, despite the fact that sterol-degrading rhodococci and mycobacteria are of great industrial and pharmaceutical interest (6,9,12,17,25,32). In recent years, interest in steroid catabolic enzymes has gained momentum, following the discovery of cholesterol catabolic gene clusters in R. jostii RHA1 and in the human pathogen Mycobacterium tuberculosis H37Rv (33). Interestingly, kshA and kshB have been identified as essential factors in the pathogenesis of M. tuberculosis H37Rv (10).KSH is a two-component enzyme system, consisting of a terminal oxygenase, KshA, and a ferredoxin reductase, KshB. The kshA an...

Gene Cluster Encoding Cholate Catabolism in Rhodococcus spp

“…Our finding that the kshA3 gene in cluster 3 is essential for growth on cholate is consistent with investigation of the orthologous gene, kshA1, in Rhodococcus rhodochrous DSM43269 (23). Five kshA paralogs from R. rhodochrous were individually tested by a complementation assay, and only kshA1 DSM43269 was able to support cholate catabolism.…”

Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons

“…Moreover, the kshB ‐ mutant failed to cleave the side‐chain of sterols and, although phytosterols were oxidized to their stenone derivatives, they were not metabolized further, suggesting that kshB could be also involved in the degradation of sterol side‐chain in Rhodococcus (van der Geize et al ., 2002a). The situation can be more complex taking into account that some strains of Rhodococcus may contain up to five KshA homologous proteins, each displaying unique steroid induction patterns and substrate ranges, confirmed that the 9α‐hydroxylation can take place at different steps of steroid oxidation (Petrusma et al ., 2011). …”

Activity of 3-Ketosteroid 9α-Hydroxylase (KshAB) Indicates Cholesterol Side Chain and Ring Degradation Occur Simultaneously in Mycobacterium tuberculosis

“…Most of the above-mentioned residues are not conserved in KshAs from Rhodococcus erythropolis SQ1, Rhodococcus rhodochrous DSM43269, and R. jostii RHA1, all of which are predicted to be involved in steroid catabolism (20,32,33). However, the substrate specificities of the rhodococcal enzymes have not been defined, and it is likely that many do not act on CoA thioester substrates.…”