期刊
BIOCHEMISTRY
卷 46, 期 31, 页码 9058-9067出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi700688x
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资金
- NCRR NIH HHS [RR021008, S10 RR021008-01, S10 RR021008, S10 RR021008-010001] Funding Source: Medline
- NIAID NIH HHS [HHSN266200400091C, AI065987, AI065251, R21 AI065261-01A1, R21 AI065261] Funding Source: Medline
- PHS HHS [HHSN266200400091C] Funding Source: Medline
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 3 beta-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 3 beta-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 (k(cat)/K-m) of 3.2 +/- 0.4 and 3.9 +/- 0.9 mu M-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 K-i of 197 +/- 8 mu M. The expression of the 3 beta-hydroxysteroid dehydrogenase in Mtb is intracellular. Disruption of the 3 beta-hydroxysteroid dehydrogenase gene in Mtb abrogates mycobacterial cholesterol oxidation activity. These data are consistent with the Rv1106c gene being the one responsible for 3 beta-hydroxysterol oxidation in Mtb.
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