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Biochem. J. (2008) 410 (339–346) (Printed in Great Britain)

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3-Keto-5a-steroid D¹-dehydrogenase from Rhodococcus erythropolis SQ1 and its orthologue in Mycobacterium tuberculosis H37Rv are highly specific enzymes that function in cholesterol catabolism

Jan KNOL, Karin BODEWITS, Gerda I. HESSELS, Lubbert DIJKHUIZEN¹ and Robert VAN DER GEIZE

Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands

The Rhodococcus erythropolis SQ1 kstD3 gene was cloned, heterologously expressed and biochemically characterized as a KSTD3 (3-keto-5a-steroid D¹-dehydrogenase). Upstream of kstD3, an ORF (open reading frame) with similarity to D⁴ KSTD (3-keto-5a-steroid D⁴-dehydrogenase) was found, tentatively designated kst4D. Biochemical analysis revealed that the D¹ KSTD3 has a clear preference for 3-ketosteroids with a saturated A-ring, displaying highest activity on 5a-AD (5a-androstane-3,17-dione) and 5a-T (5a-testosterone; also known as 17b-hydroxy-5a-androstane-3-one). The KSTD1 and KSTD2 enzymes, on the other hand, clearly prefer (9a-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, D¹ KSTD3 from R. erythropolis SQ1 clustered with Rv3537, the only D¹ 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 D¹ KSTD3, indicating that these are orthologous enzymes. The results imply that 5a-AD and 5a-T are newly identified intermediates in the cholesterol catabolic pathway, and important steroids with respect to pathogenicity.

Key words: actinomycete, 5a-androstane-3,17-dione (5a-AD), cholesterol degradation, 3-ketosteroid dehydrogenase, Rhodococcus, steroid.

Abbreviations used: ADD, 1,4-androstadiene-3,17-dione; 1-(5a)-AD, 1-(5a)-androstene-3,17-dione; 5a-AD, 5a-androstane-3,17-dione; 4-AD, 4-androstene-3,17-dione; 5a-P, 5a-pregnane-3,20-dione; 5a-T, 5a-testosterone; DCPIP, 2,6-dichlorophenol-indophenol; D⁴ KSTD, 3-keto-5a-steroid D⁴-dehydrogenase; Frd, fumarate reductase; IPTG, isopropyl b-D-thiogalactoside; KSTD, 3-ketosteroid D¹-dehydrogenase; KSTD3, 3-keto-5a-steroid D¹-dehydrogenase; LB, Luria–Bertani; NBT, Nitro Blue Tetrazolium; 9OHAD, 9a-hydroxy-4-androstene-3,17-dione; ORF, open reading frame.

¹To whom correspondence should be addressed (email L.Dijkhuizen@rug.nl).

The nucleotide sequence reported will appear in GenBank®, EMBL, DDBJ and GSBD Nucleotide Sequence Databases under the accession number EU014895.

Received 17 August 2007/7 November 2007; accepted 21 November 2007

Published as BJ Immediate Publication 21 November 2007, doi:10.1042/BJ20071130

© The Authors Journal compilation © 2008 Biochemical Society