Molecular and functional characterization of kshA and kshB, encoding two components of 3-ketosteroid 9alpha-hydroxylase, a class IA monooxygenase, in Rhodococcus erythropolis strain SQ1.
9 alpha-Hydroxylation of 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) is catalysed by 3-ketosteroid 9 alpha-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) 9 alpha-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 9 alpha-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) 9 alpha-hydroxylation. Strain RG4 was also impaired in sterol degradation, suggesting a dual role for KshB in both sterol and steroid degradation.
Department of Microbiology, Groningen Bimolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
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R
van der Geize
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Hessels
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R
van Gerwen
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P
van der Meijden
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L
Dijkhuizen
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers; D017353:Gene Deletion; D005816:Genetic Complementation Test; D006900:Hydroxylation; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D017931:DNA Primers; D007527:Isoenzymes