3-Keto-5α-steroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1 and its orthologue in Mycobacterium tuberculosis H37Rv are highly specific enzymes that function in cholesterol catabolism
Résumé
The Rhodococcus erythropolis SQ1 kstD3 gene was cloned, heterologously expressed and biochemically characterized as a 3-keto-5α-steroid Δ1-dehydrogenase (KSTD3). Upstream of kstD3, an ORF with similarity to 3-keto-5α-steroid Δ4-dehydrogenases (Δ4 KSTD) 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-androstane-3,17-dione and 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 to that of Δ1 KSTD3, indicating that these are orthologous enzymes. The results imply that 5α-androstane-3,17-dione and 17²-hydroxy-5α-androstane-3-one are newly identified intermediates in the cholesterol catabolic pathway, and important steroids with respect to pathogenicity.
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