Identification, characterization, and azole-binding properties of Mycobacterium smegmatis CYP164A2, a homolog of ML2088, the sole cytochrome P450 gene of Mycobacterium leprae

Abstract

The genome sequence of Mycobacterium leprae revealed a single open reading frame, ML2088 (CYP164A1), encoding a putative full-length cytochrome P450 monooxygenase and 12 pseudogenes. We have identified a homolog of ML2088 in Mycobacterium smegmatis and report here the cloning, expression, purification, and azole-binding characteristics of this cytochrome P450 (CYP164A2). CYP164A2 is 1,245 bp long and encodes a protein of 414 amino acids and molecular mass of 45 kDa. CYP164A2 has 60% identity with Mycobacterium leprae CYP161A1 and 66 to 69% identity with eight other mycobacterial CYP164A1 homologs, with three identified highly conserved motifs. Recombinant CYP164A2 has the typical spectral characteristics of a cytochrome P450 monooxygenase, predominantly in the ferric low-spin state. Unusually, the spin state was readily modulated by increasing ionic strength at pH 7.5, with 50% high-spin occupancy achieved with 0.14 M NaCl. CYP164A2 bound clotrimazole, econazole, and miconazole strongly (K(d), 1.2 to 2.5 muM); however, strong binding with itraconazole, ketoconazole, and voriconazole was only observed in the presence of 0.5 M NaCl. Fluconazole did not bind to CYP164A2 at pH 7.5 and no discernible type II binding spectrum was observed.

FIG. 1.

Absolute and reduced carbon monoxide difference spectra. Absolute spectra of M. smegmatis CYP51 (A) and CYP164A2 (B) were determined for the oxidized form (line 1), dithionite reduced form (line 2), and reduced carbon monoxide form (line 3). (C) Reduced carbon monoxide difference spectra were determined independently using M. smegmatis CYP51 (solid line) and CYP164A2 (dashed line) as previously described by Estabrook et al. (12) and an extinction coefficient of 91 mM⁻¹ cm⁻¹ at 448 nm (27). All spectral determinations were made using Ni²⁺-NTA-agarose-purified CYP51 (0.244 mg of protein ml⁻¹) and CYP164A2 (0.140 mg of protein ml⁻¹).

FIG. 2.

Modulation of CYP164A2 spin state by NaCl. The 100% low spin state of CYP164A2 (line 1) was obtained in the presence of 60% (vol/vol) ethylene glycol, 50 mM Tris-HCl, pH 8.2. The absolute spectrum of CYP164A2 was determined at pH 7.5 in the absence of NaCl (line 2) and in the presence of 0.5 M NaCl (line 3) and 4 M NaCl (line 4). The γ-Soret peak region (310 to 460 nm) of the spectrum has been expanded to clearly show the change in spin state from low spin (417 nm) to high spin (393 nm) caused by the increasing NaCl concentration.

FIG. 3.

Spectral titration of clotrimazole and voriconazole against M. smegmatis CYP51 and CYP164A2. Azole was progressively titrated against Ni²⁺-NTA-agarose-purified CYP51 (2.44 μM) and CYP164A2 (2.62 μM) to a maximum DMSO concentration of 2.5% (vol/vol) with the spectral difference (ΔA_peak-trough) determined after each incremental addition of azole. The type II binding spectra obtained using 4.4 μM clotrimazole (A) and 21.5 μM voriconazole (C) for both CYP51 (solid lines) and CYP164A2 (dashed lines) are shown as well as the saturation curves obtained using clotrimazole (B) and voriconazole (D) for CYP51 (hollow circles) and CYP164A2 (filled circles). Nonlinear regression (Levenberg-Marquardt algorithm) of the Hill equation, ΔA = ΔA_max/(1 + K_d/[azole]ⁿ) (28) was used to analyze the data.