The Rhodococcus erythropolis SQ1 kstD3 gene was cloned, heterologously expressed and biochemically characterized as a KSTD3 (3-keto-5α-steroid Δ1-dehydrogenase). Upstream of kstD3, an ORF (open reading frame) with similarity to Δ4 KSTD (3-keto-5α-steroid Δ4-dehydrogenase) was found, tentatively designated kst4D. Biochemical analysis revealed that the Δ1 KSTD3 has a clear preference for 3-ketosteroids with a saturated A-ring, displaying highest activity on 5α-AD (5α-androstane-3,17-dione) and 5α-T (5α-testosterone; also known as 17β-hydroxy-5α-androstane-3-one). The KSTD1 and KSTD2 enzymes, on the other hand, clearly prefer (9α-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, Δ1 KSTD3 from R. erythropolis SQ1 clustered with Rv3537, the only Δ1 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 Δ1 KSTD3, indicating that these are orthologous enzymes. The results imply that 5α-AD and 5α-T are newly identified intermediates in the cholesterol catabolic pathway, and important steroids with respect to pathogenicity.

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