Rv1106c from Mycobacterium tuberculosis is a 3beta-hydroxysteroid dehydrogenase.
New approaches are required to combat Mycobacterium tuberculosis (Mtb), especially the multi-drug resistant and extremely drug resistant organisms (MDR-TB and XDR-TB). There are many reports that mycobacteria oxidize 3beta-hydroxysterols to 3-ketosteroids, but the enzymes responsible for this activity have not been identified in mycobacterial species. In this work, the Rv1106c gene that is annotated as a 3beta-hydroxysteroid dehydrogenase in Mtb has been cloned and heterologously expressed. The purified enzyme was kinetically characterized and found to have a pH optimum between 8.5 and 9.5. The enzyme, which is a member of the short chain dehydrogenase superfamily, uses NAD+ as a cofactor and oxidizes cholesterol, pregnenolone, and dehydroepiandrosterone to their respective 3-keto-4-ene products. The enzyme forms a ternary complex with NAD+ binding before the sterol. The enzyme shows no substrate preference for dehydroepiandrosterone versus pregnenolone with second-order rate constants (kcat/Km) of 3.2 +/- 0.4 and 3.9 +/- 0.9 microM-1 min-1, respectively, at pH 8.5, 150 mM NaCl, 30 mM MgCl2, and saturating NAD+. Trilostane is a competitive inhibitor of dehydroepiandrosterone with a Ki of 197 +/- 8 microM. The expression of the 3beta-hydroxysteroid dehydrogenase in Mtb is intracellular. Disruption of the 3beta-hydroxysteroid dehydrogenase gene in Mtb abrogates mycobacterial cholesterol oxidation activity. These data are consistent with the Rv1106c gene being the one responsible for 3beta-hydroxysterol oxidation in Mtb.
Yang
Xinxin
X
Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA.
Dubnau
Eugenie
E
Smith
Issar
I
Sampson
Nicole S
NS
D015096:3-Hydroxysteroid Dehydrogenases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism; D019295:Computational Biology; D003687:Dehydroepiandrosterone / Q000737:chemistry / Q000378:metabolism; D003902:Detergents / Q000737:chemistry / Q000378:metabolism; D013196:Dihydrotestosterone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D009994:Osmolar Concentration; D010802:Phylogeny; D011284:Pregnenolone / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry; D013261:Sterols / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003902:Detergents; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013261:Sterols; D013196:Dihydrotestosterone; D009243:NAD; D003687:Dehydroepiandrosterone; D011284:Pregnenolone; D002784:Cholesterol; D015096:3-Hydroxysteroid Dehydrogenases; C009954:trilostane