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

Abstract

The Rhodococcus erythropolis SQ1 kstD3 gene was cloned, heterologously expressed and biochemically characterized as a KSTD3 (3-keto-5alpha-steroid Delta(1)-dehydrogenase). Upstream of kstD3, an ORF (open reading frame) with similarity to Delta(4) KSTD (3-keto-5alpha-steroid Delta(4)-dehydrogenase) was found, tentatively designated kst4D. Biochemical analysis revealed that the Delta(1) KSTD3 has a clear preference for 3-ketosteroids with a saturated A-ring, displaying highest activity on 5alpha-AD (5alpha-androstane-3,17-dione) and 5alpha-T (5alpha-testosterone; also known as 17beta-hydroxy-5alpha-androstane-3-one). The KSTD1 and KSTD2 enzymes, on the other hand, clearly prefer (9alpha-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, Delta(1) KSTD3 from R. erythropolis SQ1 clustered with Rv3537, the only Delta(1) 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 Delta(1) KSTD3, indicating that these are orthologous enzymes. The results imply that 5alpha-AD and 5alpha-T are newly identified intermediates in the cholesterol catabolic pathway, and important steroids with respect to pathogenicity.

Characterization of the KstR-dependent promoter of the gene for the first step of the cholesterol degradative pathway in Mycobacterium smegmatis

“…Interestingly, it has been described that only fast-growing non-pathogenic mycobacteria were able to grow on cholesterol as a sole carbon source (Av-Gay & Sobouti, 2000); however, more recent results have demonstrated that cholesterol is also used as a carbon source during infection of M. tuberculosis (Pandey & Sassetti, 2008). Although recent biochemical and structural studies have assigned a role for some of the mycobacterial genes in cholesterol catabolism, the complete degradative pathway and its specific regulation remain to be fully established (Capyk et al, 2009;Knol et al, 2008;Lack et al, 2010;Yam et al, 2009). Recently, it has been shown that cholesterol utilization in mycobacteria is controlled by two TetR-type transcriptional repressors (Kendall et al, 2007(Kendall et al, , 2010.…”

Purification, crystallization and preliminary X-ray crystallographic analysis of 3-ketosteroid Δ1-dehydrogenase fromRhodococcus erythropolisSQ1

“…Total DNA from R. erythropolis SQ1 has been isolated (van der Geize et al, 2000) and characterized to contain three genes, kstD1 (van der Geize et al, 2001), kstD2 (van der Geize et al, 2002) and kstD3 (Knol et al, 2008), that code for three different 3-ketosteroid Á 1 -dehydrogenases. The kstD1 gene (1533 bp; GenBank accession No.…”

“…The activity of the dehydrogenase is dependent on FAD (flavin adenine dinucleotide) and requires the presence of a carbonyl group at the C3 position of the steroid substrate (Itagaki, Matushita et al, 1990;Itagaki, Wakabayashi et al, 1990). The enzyme acts on a variety of 3-ketosteroid substrates, with a preference for substrates possessing a double bond at the C4-C5 position (Itagaki, Wakabayashi et al, 1990;Knol et al, 2008) such as, for example, the main catabolic steroid intermediate 4-androstene-3,17-dione (Fig. 1).…”

“…erythropolis SQ1 has three 3-ketosteroid Á 1 -dehydrogenase isoenzymes: Á 1 -KSTD1 (van der Geize et al, 2001), Á 1 -KSTD2 (van der Geize et al, 2002) and Á 1 -KSTD3 (Knol et al, 2008). Á 1 -KSTD1 has been expressed in Escherichia coli strain BL21 (DE3) at higher levels than was possible with the other two isoenzymes and, in contrast to the latter isoenzymes, could be purified relatively easily (Knol et al, 2008).…”

“…erythropolis SQ1 has three 3-ketosteroid Á 1 -dehydrogenase isoenzymes: Á 1 -KSTD1 (van der Geize et al, 2001), Á 1 -KSTD2 (van der Geize et al, 2002) and Á 1 -KSTD3 (Knol et al, 2008). Á 1 -KSTD1 has been expressed in Escherichia coli strain BL21 (DE3) at higher levels than was possible with the other two isoenzymes and, in contrast to the latter isoenzymes, could be purified relatively easily (Knol et al, 2008). Amino-acid sequence alignment of Á 1 -KSTD1 with the sequences of structurally characterized enzymes showed that Á 1 -KSTD1 has the highest homology to 3-ketosteroid Á 4 -(5 )-dehydrogenase from R. jostii (van Oosterwijk et al, unpublished work) followed by flavocytochrome c fumarate reductase from Shewanella putrefaciens (PDB entry 1d4c; Leys et al, 1999), with sequence identities of 28 and 24%, respectively.…”