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Microbiology 148 (2002), 3285-3292
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Microbiology (2002), 148, 3285-3292.
© 2002 Society for General Microbiology

Research Paper

Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid {Delta}1-dehydrogenase isoenzymeb

Robert van der Geize1, Gerda I. Hessels1 and Lubbert Dijkhuizen1

Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands1

Author for correspondence: Lubbert Dijkhuizen. Tel: +31 50 3632153. Fax: +31 50 3632154. e-mail: L.Dijkhuizen{at}biol.rug.nl

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 9{alpha}-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.

Keywords: 3-ketosteroid dehydrogenase, isoenzymes, unmarked gene deletion, steroid

Abbreviations: AD, 4-androstene-3,17-dione; ADD, 1,4-androstadiene-3,17-dione; DCPIP, 2,6-dichlorophenolindophenol; KSH, 3-ketosteroid 9{alpha}-hydroxylase; KSTD, 3-ketosteroid {Delta}1-dehydrogenase; 9OHAD, 9{alpha}-hydroxy-4-androstene-3,17-dione; PMS, phenazine methosulphate

b The GenBank accession number for the sequence reported in this paper is AY078169




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