Rv1106c from Mycobacterium tuberculosis is a 3beta-hydroxysteroid dehydrogenase

Abstract

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.

Figure 1

Unrooted phylogenetic tree for 3β-hydroxysteroid dehydrogenase encoded by Rv1106c and related proteins. The values are relative evolutionary distance. The tree was generated using a ClustalW 1.82 alignment and CLC Free Workbench 3. Proteins are identified by species, enzyme, and Uniprot ID.

Figure 2

pH dependence of the reaction catalyzed by rH₆3BHSD with DHEA as the varied substrate. Conditions: 2.8 mM NAD⁺, 100 mM TAPS hydrochloride buffer, 150 mM NaCl, 30 mM MgCl₂, pH 8.5, 30 °C. A, pH dependence of k_cat. B, pH dependence of k_ca/K_m. The curves are fits to eq. (7). The data shown are the average of three independent experiments, and the errors are the standard deviation of measurement.

Figure 2

pH dependence of the reaction catalyzed by rH₆3BHSD with DHEA as the varied substrate. Conditions: 2.8 mM NAD⁺, 100 mM TAPS hydrochloride buffer, 150 mM NaCl, 30 mM MgCl₂, pH 8.5, 30 °C. A, pH dependence of k_cat. B, pH dependence of k_ca/K_m. The curves are fits to eq. (7). The data shown are the average of three independent experiments, and the errors are the standard deviation of measurement.

Figure 3

Cation dependence of rH₆3BHSD activity. A, Specific activity in the presence of assorted cations or EDTA. Conditions: 150 μM DHEA, 3.5 mM NAD⁺, 100 mM triethanolamine hydrochloride buffer, pH 8.5, 30 °C. B, Specific activity as a function of NaCl concentration. Conditions: 150 μM DHEA, 2.8 mM NAD⁺, 100 mM TAPS hydrochloride buffer, pH 8.5, 150 mM NaCl, 30 °C. C, Specific activity in the presence of 150 mM NaCl and assorted cations or EDTA. Conditions: 150 μM DHEA, 2.8 mM NAD⁺, 100 mM TAPS hydrochloride buffer, pH 8.5, 150 mM NaCl, 30 °C. The data shown are the average of three independent experiments, and the errors are the standard deviation of measurement.

Figure 3

Cation dependence of rH₆3BHSD activity. A, Specific activity in the presence of assorted cations or EDTA. Conditions: 150 μM DHEA, 3.5 mM NAD⁺, 100 mM triethanolamine hydrochloride buffer, pH 8.5, 30 °C. B, Specific activity as a function of NaCl concentration. Conditions: 150 μM DHEA, 2.8 mM NAD⁺, 100 mM TAPS hydrochloride buffer, pH 8.5, 150 mM NaCl, 30 °C. C, Specific activity in the presence of 150 mM NaCl and assorted cations or EDTA. Conditions: 150 μM DHEA, 2.8 mM NAD⁺, 100 mM TAPS hydrochloride buffer, pH 8.5, 150 mM NaCl, 30 °C. The data shown are the average of three independent experiments, and the errors are the standard deviation of measurement.

Figure 3

Cation dependence of rH₆3BHSD activity. A, Specific activity in the presence of assorted cations or EDTA. Conditions: 150 μM DHEA, 3.5 mM NAD⁺, 100 mM triethanolamine hydrochloride buffer, pH 8.5, 30 °C. B, Specific activity as a function of NaCl concentration. Conditions: 150 μM DHEA, 2.8 mM NAD⁺, 100 mM TAPS hydrochloride buffer, pH 8.5, 150 mM NaCl, 30 °C. C, Specific activity in the presence of 150 mM NaCl and assorted cations or EDTA. Conditions: 150 μM DHEA, 2.8 mM NAD⁺, 100 mM TAPS hydrochloride buffer, pH 8.5, 150 mM NaCl, 30 °C. The data shown are the average of three independent experiments, and the errors are the standard deviation of measurement.

Figure 4

Steady state kinetics of rHg₆BHSD. A plot of initial velocity against NAD⁺ at fixed DHEA was fitted to equation (5). Conditions: 120 μM DHEA, 100 mM TAPS hydrochloride buffer, 150 mM NaCl, 30 mM MgCl₂, pH 8.5, 30 °C. The data shown are the average of three independent experiments, and the errors are the standard deviation of measurement.

Scheme 1

Reaction catalyzed by Mtb 3β-hydroxy steroid dehydrogenase (Rv1106c).