3-Keto-5alpha-steroid Delta(1)-dehydrogenase from Rhodococcus erythropolis SQ1 and its orthologue in Mycobacterium tuberculosis H37Rv are highly specific enzymes that function in cholesterol catabolism.
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.
Knol
Jan
J
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands.
Bodewits
Karin
K
Hessels
Gerda I
GI
Dijkhuizen
Lubbert
L
van der Geize
Robert
R
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015698:Genomic Library; D007664:Ketosteroids / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D007664:Ketosteroids; D011994:Recombinant Proteins; D002784:Cholesterol; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase