1887

Abstract

Previously, SQ1 , encoding ketosteroid Δ-dehydrogenase (KSTD1) was characterized. Surprisingly, a 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 Δ-dehydrogenation of 4-androstene-3,17-dione (AD) and 9α-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 () showing clear similarity (35% identity at amino acid sequence level) with KSTD1. Expression of in resulted in high KSTD2 activity levels. Single gene deletion mutants of either (strain RG1) or (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 double deletion mutant (strain RG8) was constructed. Strain RG8 was unable to grow on steroid substrates, had undetectable steroid Δ-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.

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2002-10-01
2019-10-15
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