Gerda Hessels

Molecular and functional characterization of kshA and kshB, encoding two components of 3-ketosteroid 9α-hydroxylase, a class IA monooxygenase, in Rhodococcus erythropolis strain SQ1

9α‐Hydroxylation of 4‐androstene‐3,17‐dione (AD) and 1,4‐androstadiene‐3,17‐dione (ADD) is catalysed by 3‐ketosteroid 9α‐hydroxylase (KSH), a key enzyme in microbial steroid catabolism. Very limited knowledge is presently available on the KSH enzyme. Here, we report for the first time the identification and molecular characterization of genes encoding KSH activity. The kshA and kshB genes, encoding KSH in Rhodococcus erythropolis strain SQ1, were cloned by functional complementation of mutant strains blocked in AD(D) 9α‐hydroxylation. Analysis of the deduced amino acid sequences of kshA and kshB showed that they contain domains typically conserved in class IA terminal oxygenases and class IA oxygenase reductases respectively. By definition, class IA oxygenases are made up of two components, thus classifying the KSH enzyme system in R. erythropolis strain SQ1 as a two‐component class IA monooxygenase composed of KshA and KshB. Unmarked in frame gene deletion mutants of parent strain R. erythropolis SQ1, designated strains RG2 (kshA mutant) and RG4 (kshB mutant), were unable to grow on steroid substrates AD(D), whereas growth on 9α‐hydroxy‐4‐androstene‐3,17‐dione (9OHAD) was not affected. Incubation of these mutant strains with AD resulted in the accumulation of ADD (30–50% conversion), confirming the involvement of KshA and KshB in AD(D) 9α‐hydroxylation. Strain RG4 was also impaired in sterol degradation, suggesting a dual role for KshB in both sterol and steroid degradation.

Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid Delta(1)-dehydrogenase isoenzyme

Previously, Rhodococcus erythropolis SQ1 kstD, encoding ketosteroid Delta(1)-dehydrogenase (KSTD1) was characterized. Surprisingly, a kstD gene deletion mutant (strain RG1) grew normally on steroids. UV mutagenesis of strain RG1 allowed isolation of strains (e.g. strain RG1-UV29) unable to perform the Delta(1)-dehydrogenation of 4-androstene-3,17-dione (AD) and 9alpha-hydroxy-4-androstene-3,17-dione (9OHAD). Functional complementation of strain RG1-UV29 with total genomic DNA of strain RG1 resulted in identification of a 1698 nt ORF (kstD2) showing clear similarity (35% identity at amino acid sequence level) with KSTD1. Expression of kstD2 in Escherichia coli resulted in high KSTD2 activity levels. Single gene deletion mutants of either kstD (strain RG1) or kstD2 (strain RG7) appeared unaffected in growth on the steroid substrates AD, 1,4-androstadiene-3,17-dione and 9OHAD. Strain RG7, but not strain RG1, showed temporary accumulation of 9OHAD during AD conversion. A kstD kstD2 double deletion mutant (strain RG8) was constructed. Strain RG8 was unable to grow on steroid substrates, had undetectable steroid Delta(1)-dehydrogenation activity and efficiently converted AD into 9OHAD. Strain SQ1 thus employs two KSTD isoenzymes in steroid catabolism. Analysis of two null mutants in KSTD2 showed that the semi-conserved Ser325 and the highly conserved Thr503 play a role in KSTD enzyme activity.

3-Keto-5 alpha-steroid Delta'-dehydrogenase from Rhodococcus erythropolis SQ1 and its orthologue in Mycobacterium tuberculosis H37Rv are highly specific enzymes that function in cholesterol catabolism

The Rhodococcus erythropolis SQ1 kstD3 gene was cloned, heterologously expressed and biochemically characterized as a KSTD3 (3-keto-5alpha-steroid Delta(1)-dehydrogenase). Upstream of kstD3, an ORF (open reading frame) with similarity to Delta(4) KSTD (3-keto-5alpha-steroid Delta(4)-dehydrogenase) was found, tentatively designated kst4D. Biochemical analysis revealed that the Delta(1) KSTD3 has a clear preference for 3-ketosteroids with a saturated A-ring, displaying highest activity on 5alpha-AD (5alpha-androstane-3,17-dione) and 5alpha-T (5alpha-testosterone; also known as 17beta-hydroxy-5alpha-androstane-3-one). The KSTD1 and KSTD2 enzymes, on the other hand, clearly prefer (9alpha-hydroxy-)4-androstene-3,17-dione as substrates. Phylogenetic analysis of known and putative KSTD amino acid sequences showed that the R. erythropolis KSTD proteins cluster into four distinct groups. Interestingly, Delta(1) KSTD3 from R. erythropolis SQ1 clustered with Rv3537, the only Delta(1) KSTD present in Mycobacterium tuberculosis H37Rv, a protein involved in cholesterol catabolism and pathogenicity. The substrate range of heterologously expressed Rv3537 enzyme was nearly identical with that of Delta(1) KSTD3, indicating that these are orthologous enzymes. The results imply that 5alpha-AD and 5alpha-T are newly identified intermediates in the cholesterol catabolic pathway, and important steroids with respect to pathogenicity.

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

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 9α-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 α-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 α-hydroxylation occurring at different levels during microbial steroid degradation.

Identification of the minimal replicon of plasmid pMEA300 of the methylotrophic actinomycete Amycolatopsis methanolica

The actinomycete Amycolatopsis methanolica contains a 13.3 kb plasmid (pMEA300), capable of enhancing the spontaneous mutation frequency of its host. Depending on the growth medium pMEA300 is not only maintained as an integrated element but can additionally be present as a multicopy, autonomously replicating plasmid. The minimal replicon of pMEA300 was identified. Two unlinked DNA fragments of 2.6 kb and 0.8 kb were required for pMEA300 maintenance. Sequence analysis of the 2.6 kb fragment revealed at least two open reading frames, orfA and orfB, encoding putative proteins of 170 amino acids (18 373 Da) and 416 amino acids (45 260 Da), respectively. No clear similarities were found between the deduced amino acid sequences of the putative orfA and orfB products of pMEA300 and replication proteins identified for various Streptomyces plasmids. The pMEA300 proteins of A. methanolica thus may represent unfamiliar types. The 0.8 kb fragment contained a single complete open reading frame (korA), encoding a protein of 118 amino acids (12 917 Da). The putative KorA protein of pMEA300 shows sequence similarity with various other Streptomyces plasmid‐encoded Kor proteins which may belong to the GntR family of transcriptional repressor proteins. The data provide preliminary evidence for the possible involvement of a kil—kor system in autonomous replication of pMEA300.