This paper reports the biochemical characterization of a purified and reconstituted two-component 3-ketosteroid 9␣-hydroxylase (KSH). KSH of Rhodococcus rhodochrous DSM 43269, consisting of a ferredoxin reductase (KshB) and a terminal oxygenase (KshA), was heterologously expressed in Escherichia coli. E. coli cell cultures, expressing both KshA and KshB, converted 4-androstene-3,17-dione (AD) into 9␣-hydroxy-4-AD (9OHAD) with a >60% molar yield over 48 h of incubation. Coexpression and copurification were critical to successfully obtain pure and active KSH. Biochemical analysis revealed that the flavoprotein KshB is an NADH-dependent reductase using flavin adenine dinucleotide as a cofactor. Reconstitution experiments confirmed that KshA, KshB, and NADH are essential for KSH activity with steroid substrates. KSH hydroxylation activity was inhibited by several divalent metal ions, especially by zinc. The reconstituted KSH displayed subtle steroid substrate specificity; a range of 3-ketosteroids, i.e., 5␣-⌯, 5-⌯, ⌬1, and ⌬4 steroids, could act as KSH substrates, provided that they had a short side chain. The formation of 9OHAD from AD by KSH was confirmed by liquid chromatography-mass spectrometry analysis and by the specific enzymatic conversion of 9OHAD into 3-hydroxy-9,10-secoandrost-1,3,5(10)-triene-9,17-dione using 3-ketosteroid ⌬1-dehydrogenase. Only a single KSH is encoded in the genome of the human pathogen Mycobacterium tuberculosis H37Rv, shown to be important for survival in macrophages. Since no human KSH homolog exists, the M. tuberculosis enzyme may provide a novel target for treatment of tuberculosis. Detailed knowledge about the biochemical properties of KSH thus is highly relevant in the research fields of biotechnology and medicine.Hydroxylated steroids are pharmaceutically very interesting bioactive compounds. 9␣-Hydroxylated steroids are of particular importance for the synthesis of corticoids such as 9␣-fluorohydrocortisone. Microorganisms are widely used for the stereo-specific hydroxylation of steroids, but little is known about the enzymes involved, and current processes suffer from low conversion rates and yields (12,18,23).Rhodococcus species are well known for their broad catabolic potential and ability to degrade sterols and steroids (14,21,25,39). In this paper, we focus on 3-ketosteroid 9␣-hydroxylase (KSH), which is essential for the growth of Rhodococcus strains on steroids (38). KSH acts on the B-ring of 3-keto-⌬4 steroids, e.g., 4-androstene-3,17-dione (AD), introducing a 9␣-hydroxyl moiety (Fig. 1). Subsequent ⌬1-dehydrogenation of 9␣-hydroxy-AD (9OHAD) by 3-ketosteroid ⌬1-dehydrogenase (⌬1-KSTD) initiates the opening of the B-ring through formation of a chemically unstable intermediate that spontaneously hydrolyzes, forming 3-hydroxy-9,10-secoandrost-1,3,5(10)-triene-9,17-dione (3-HSA). KSH activity has been observed in various actinobacterial genera, e.g., Mycobacterium (1,3,6), Nocardia (35), Arthrobacter (11), and Rhodococcus (38). In view of their amino acid sequences, KSH ...
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...
Exonization of retroposed mobile elements, a process whereby new exons are generated following changes in non-protein-coding regions of a gene, is thought to have great potential for generating proteins with novel domains. Our previous analysis of primate-specific Alu-short interspersed elements (SINEs) showed, however, that during their 60 million years of evolution, SINE exonizations occurred in some primates, only to be lost again in some of the descendent lineages. This dynamic gain and loss makes it difficult to ascertain the contribution of exonization to genomic novelty. It was speculated that Alu-SINEs are too young to reveal persistent protein exaptation. In the present study we examined older mobile elements, mammalian-wide interspersed repeats (MIRs) that underwent active retroposition prior to the placental mammalian radiation ∼130 million years ago, to determine their contribution to protein-coding sequences. Of 107 potential cases of MIR exonizations in human, an analysis of splice sites substantiates a mechanism that benefits from 3Ј splice site selection in MIR sequences. We retraced in detail the evolution of five MIR elements that exonized at different times during mammalian evolution. Four of these are expressed as alternatively spliced transcripts; three in species throughout the mammalian phylogenetic tree and one solely in primates. The fifth is the first experimentally verified, constitutively expressed retroposed SINE element in mammals. This pattern of highly conserved, alternatively and constitutively spliced MIR sequences evinces the potential of exonized transposed elements to evolve beyond the transient state found in Alu-SINEs and persist as important parts of functional proteins.
The actinobacterial cholesterol catabolic gene cluster contains a subset of genes that encode β-oxidation enzymes with a putative role in sterol side chain degradation. We investigated the physiological roles of several genes, i.e., fadD17, fadD19, fadE26, fadE27, and ro04690DSM43269, by gene inactivation studies in mutant strain RG32 of Rhodococcus rhodochrous DSM43269. Mutant strain RG32 is devoid of 3-ketosteroid 9α-hydroxylase (KSH) activity and was constructed following the identification, cloning, and sequential inactivation of five kshA gene homologs in strain DSM43269. We show that mutant strain RG32 is fully blocked in steroid ring degradation but capable of selective sterol side chain degradation. Except for RG32ΔfadD19, none of the mutants constructed in RG32 revealed an aberrant phenotype on sterol side chain degradation compared to parent strain RG32. Deletion of fadD19 in strain RG32 completely blocked side chain degradation of C-24 branched sterols but interestingly not that of cholesterol. The additional inactivation of fadD17 in mutant RG32ΔfadD19 also did not affect cholesterol side chain degradation. Heterologously expressed FadD19DSM43269 nevertheless was active toward steroid-C26-oic acid substrates. Our data identified FadD19 as a steroid-coenzyme A (CoA) ligase with an essential in vivo role in the degradation of the side chains of C-24 branched-chain sterols. This paper reports the identification and characterization of a CoA ligase with an in vivo role in sterol side chain degradation. The high similarity (67%) between the FadD19(DSM43269) and FadD19H37Rv enzymes further suggests that FadD19H37Rv has an in vivo role in sterol metabolism of Mycobacterium tuberculosis H37Rv.
BackgroundBacteria of the genus Rhodococcus are well known for their ability to degrade a large range of organic compounds. Some rhodococci are free-living, saprophytic bacteria; others are animal and plant pathogens. Recently, several studies have shown that their genomes encode putative pathways for the synthesis of a large number of specialized metabolites that are likely to be involved in microbe-microbe and host-microbe interactions. To systematically explore the specialized metabolic potential of this genus, we here performed a comprehensive analysis of the biosynthetic coding capacity across publicly available rhododoccal genomes, and compared these with those of several Mycobacterium strains as well as that of their mutual close relative Amycolicicoccus subflavus.ResultsComparative genomic analysis shows that most predicted biosynthetic gene cluster families in these strains are clade-specific and lack any homology with gene clusters encoding the production of known natural products. Interestingly, many of these clusters appear to encode the biosynthesis of lipopeptides, which may play key roles in the diverse environments were rhodococci thrive, by acting as biosurfactants, pathogenicity factors or antimicrobials. We also identified several gene cluster families that are universally shared among all three genera, which therefore may have a more ‘primary’ role in their physiology. Inactivation of these clusters by mutagenesis might help to generate weaker strains that can be used as live vaccines.ConclusionsThe genus Rhodococcus thus provides an interesting target for natural product discovery, in view of its large and mostly uncharacterized biosynthetic repertoire, its relatively fast growth and the availability of effective genetic tools for its genomic modification.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3966-1) contains supplementary material, which is available to authorized users.
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