Rv1106c from Mycobacterium tuberculosis Is a 3β-Hydroxysteroid Dehydrogenase

Abstract

New approaches are required to combat M. tuberculosis (Mtb), especially the multi and extremely drug resistant and organisms (MDR-TB and XDR-TB). There are many reports that mycobacteria oxidize 3β-hydroxysterols to 3-ketosteroids, but the enzyme(s) responsible for this activity have not been identified in mycobacterial species. In this work, the Rv1106c gene that is annotated as a 3β-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 μM −1 min −1 and 3.9 ± 0.9 μM −1 min −1 , respectively at pH 8.5, 150 mM NaCl, 30 mM MgCl 2 , and saturating NAD + . Trilostane is a competitive inhibitor of dehydroepiandrosterone with a K i of 197 ± 8 |μM. The expression of the 3β-hydroxysteroid dehydrogenase in Mtb is intracellular. Disruption of the 3β-hydroxysteroid dehydrogenase gene in Mtb abrogates mycobacterial cholesterol oxidation activity. These data are consistent with Rv1106c being the gene responsible for 3β-hydroxysterol oxidation in Mtb.Tuberculosis is an opportunistic infection caused by Mycobacterium tuberculosis (Mtb 1 ) in individuals with HIV-AIDS that is estimated to infect 30% of the world's population (1,2). The World Health Organization estimates that 2 million people die every year from tuberculosis. Drug resistance to front-line Mtb drugs rifampicin and isoniazid has emerged (3,4) and additional resistance to second line drugs is emerging (5,6). It is clear that new approaches are required to combat these multi drug-resistant and extreme (or extensively) drugresistant organisms (7-9).The complete genome sequences of microorganisms are a rich source for mining new drug targets. However, oftentimes, biochemical functions have been assigned to genes purely on the basis of their sequence homology to gene products which are themselves poorly † Financial support from the National Institutes of Health (AI065251 (N.S.S.), AI065997 (I.S.), RR021008, (N.S.S.) and NIAID Contract# HHSN266200400091C, (Colorado State University) and the National Science Foundation (CHE0131146 for NMR spectrometers) is gratefully acknowledged. *corresponding author: Address: Stony Brook University, Stony Brook, New York 11794-3400, Phone: (631) Fax: (631) 632-5731 Nicole.Sampson@StonyBrook.edu. 1 Mtb: Mycobacterium tuberculosis; MDR-TB: multi-drug resistant Mycobacterium tuberculosis; XDR-TB: extremely-drug resistant Mycobacterium tuberculosis; DHEA: dehydroepiandrosterone; rH 6 3BHSD: recombinant 3β-hydroxysteroid dehydrogenase with an Nterminal six histidine tag; rSBHSD...

igr Genes and Mycobacterium tuberculosis Cholesterol Metabolism

“…Mycobacterium-dependent accumulation of cholesterol esters in macrophages has been observed (9), and recently M. tuberculosis genes were shown to encode cholesterolmodifying activities that contribute to virulence (2,19,21,22).…”

Cholesterol Analogs with Degradation-resistant Alkyl Side Chains Are Effective Mycobacterium tuberculosis Growth Inhibitors

“…This oxidation reaction can occur with cholesterol itself, but does so more efficiently with cholest-4-en-3-one (4), the metabolite formed by oxidation of the 3-hydroxyl group to a ketone by a mycobacterial 3␤-hydroxysterol dehydrogenase (3␤-HSD, Rv1106c) (11)(12)(13). The ␤-oxidation of the side chain gives rise to 4-androstenedione, which can undergo further degradation (3).…”

Cholesterol Is Not an Essential Source of Nutrition for Mycobacterium tuberculosis during Infection

“…The myc11 mutant formed colonies as readily as the mc 2 155 wild-type strain (data not shown). Complementation of the hsd mutant with the wild-type gene and 1,000 bases upstream of the open reading frame (26) completely restored growth on cholesterol (Fig. 2).…”

“…In M. tuberculosis, Rv1106c (hsd) is the closest homolog (75% identity with the Nocardia enzyme, UniProtKB ID Q03704). Indeed, we demonstrated in earlier work that Rv1106c encodes a functional 3␤-hydroxysteroid dehydrogenase (HSD) that can utilize cholesterol, pregnenolone, and dehydroepiandosterone as substrates (26). Here, we investigate the essentiality of these genes for growth of M. tuberculosis in vitro and in vivo.…”