Characterization and catalytic properties of the sterol 14alpha-demethylase from Mycobacterium tuberculosis

Abstract

Sterol 14alpha-demethylase encoded by CYP51 is a mixed-function oxidase involved in sterol synthesis in eukaryotic organisms. Completion of the Mycobacterium tuberculosis genome project revealed that a protein having homology to mammalian 14alpha-demethylases might be present in this bacterium. Using genomic DNA from mycobacterial strain H(37)Rv, we have established unambiguously that the CYP51-like gene encodes a bacterial sterol 14alpha-demethylase. Expression of the M. tuberculosis CYP51 gene in Escherichia coli yields a P450, which, when purified to homogeneity, has the predicted molecular mass, ca. 50 kDa on SDS/PAGE, and binds both sterol substrates and azole inhibitors of P450 14alpha-demethylases. It catalyzes 14alpha-demethylation of lanosterol, 24, 25-dihydrolanosterol, and obtusifoliol to produce the 8,14-dienes stereoselectively as shown by GC/MS and (1)H NMR analysis. Both flavodoxin and ferredoxin redox systems are able to support this enzymatic activity. Structural requirements of a 14alpha-methyl group and Delta(8(9))-bond were established by comparing binding of pairs of sterol substrate that differed in a single molecular feature, e.g., cycloartenol paired with lanosterol. These substrate requirements are similar to those established for plant and animal P450 14alpha-demethylases. From the combination of results, the interrelationships of substrate functional groups within the active site show that oxidative portions of the sterol biosynthetic pathway are present in prokaryotes.

Figure 1

(A) Sterol molecule structures. 1, lanosterol; 2, cycloartenol; 3, parkeol; 4, DHL; 5, zymosterol; and 6, obtusifoliol. (B) DHL 14α-demethylation. Conversion of DHL to 4,4-dimethyl-5α-cholesta-8,14-diene-3β-ol in the presence of MT P45014DM, NADPH, and molecular oxygen. (C) GLC profile of overnight conversion of 2 mg DHL. E. coli Fld/Fdr system was used as P450 electron donor. The peaks at 16.38 and 17 min correspond to the DHL and metabolite retention times, respectively.

Figure 2

(A) Potential Shine–Dalgarno sequence (shadowed box) of the MT CYP51 gene; the ATG is represented in boldface characters. (B) Absorbance of purified MT P45014DM (400 pmol), absolute oxidized form (regular trace), and sodium hydrosulfite reduced form (dashed trace). Inset shows the α- and β-bands for the oxidized and the reduced forms. (C) Differential CO-reduced P450 spectrum of purified MT P45014DM (400 pmol). (D) Silver staining (1) and immunoblot analysis (2) using 1 pmol and 0.4 pmol of purified MT P45014DM, respectively. MT P45014DM antibody prepared with TiterMax@Gold as adjuvant was used at a 1:5,000 dilution. Protein G-horseradish peroxidase conjugate (Bio-Rad) was used as a second antibody, and an enhanced chemiluminescence kit was used for detection.

Figure 3

Comparison of MT P45014DM activities supported by either Fld/Fdr or Fdx/Fnr. [24-³H]DHL was converted overnight at 30°C by 1 nmol MT P45014DM with either 20 nmol Fld and 2 nmol Fdr (A) or 20 nmol Fdx and 2 nmol Fnr (B) (30). Peaks S and P correspond to DHL and its 14α-demethylated product, respectively. Peaks U are unidentified products. MT P45014DM used in this experiment was purified further by HLPC (BIOCAD/Sprint, Perspective Biosystems, Framingham, MA) by using Poros HS and HQ columns (Perspective Biosystems). The HS flowthrough is loaded on an HQ column and eluted by using a NaCl gradient (150–500 mM).

Figure 4

(A) MT P45014DM type I binding spectrum for obtusifoliol (100 nM–5 μM). (B) Double reciprocal plot for obtusifoliol (●), DHL (▴), and lanosterol (■) binding with 10 μM MT P45014DM. (C) MT P45014DM type II binding spectrum in the presence of clotrimazole (500 nM–100 μM). (D) Double reciprocal plot for clotrimazole (○), ketoconazole (●), and fluconazole (▴) binding with 5 μM MT P45014DM.

Figure 5

Western blot analysis of 0.4 pmol purified recombinant MT P45014DM (lanes 1 and 3) and 100 μg of MT cytosolic fraction (lanes 2 and 4) by using complete (lanes 1 and 2) and depleted (lanes 3 and 4) antisera. The antiserum raised by using Freund’s adjuvant was purified using an MT P45014DM Sepharose affinity column followed by batch chromatography with the same resin (40). The antiserum was depleted by overnight incubation with 6 nmol purified MT P45014DM at 4°C.