Functional expression and characterization of CYP51 from dandruff-causing Malassezia globosa

Abstract

Malassezia globosa is one of the most common yeasts to cause various human skin diseases including dandruff and seborrheic dermatitis. Genomic analysis of M. globosa revealed four putative cytochrome P450 (CYP) enzymes. Here, we report the purification and characterization of recombinant CYP51, a putative lanosterol 14α-demethylase, from M. globosa. The M. globosa CYP51 was expressed heterologously in Escherichia coli, followed by purification. Purified CYP51 showed a typical reduced CO-difference spectrum of P450, with a maximum absorption at 447 nm. Purified CYP51 exhibited tight binding to azole antifungal agents such as ketoconazole, econazole, fluconazole, or itraconazole, with K(d) values around 0.26-0.84 μM, which suggests that CYP51 is an orthologous target for antifungal agents in the M. globosa. In addition, three mutations (Y127F, A169S, and K176N) in the amino acid sequence of M. globosa CYP51 were identified in one of the azole-resistant strains. Homology modeling of M. globosa CYP51 suggested that the Y127F mutation may influence the resistance to azoles by blocking substrate access channels. Taken together, functional expression and characterization of the CYP51 enzyme can provide a fundamental basis for a specific antifungal drug design for dandruff caused by M. globosa.

Heterologous expression and characterization of CYP61A1 from dandruff-causing Malassezia globosa

“…The binding of ketoconazole, fluconazole and itraconazole to CYP61A1 yielded K s values of 0.58, 0.69, and 1.08 lM, respectively (Table 2), indicating very tight binding to CYP61A1. These binding affinity patterns of CYP61A1 are similar to those previously reported for M. globosa CYP51 [12]. These findings suggest that the antifungal activities of azoles can, in part, be attributed to inhibition of CYP61A1 enzymatic activity during sterol biosynthesis in M. globosa.…”

“…The expression and purification of CYP61A1 were carried out as previously described with some modifications [12,24,25]. E. coli DH5a was transformed with the pCW(Mg61A1) vector and then inoculated into TB medium containing 100 lg mL À1 ampicillin, 1 mM thiamine, 0.5 mM of d-aminolevulinic acid, and 1.0 mM IPTG.…”

“…Sequence alignment analyses indicate that the M. globosa genome includes four genes coding for putative CYP enzymes, including MGL_2415, MGL_3996, MGL_1059, and MGL_0310 [11,12]. Among them, MGL_0310 encodes CYP61A1, a sterol C-22 desaturase enzyme that catalyzes a C-22 desaturation reaction for the conversion of ergosta 5, 7-dienol into ergosterol in M. globosa [13][14][15][16].…”

The Malassezia Genus in Skin and Systemic Diseases

“…The disease is more common in certain populations, such as the elderly (181), and can be severe and therapy resistant in neuroleptic-induced Parkinsonism (31) and HIV patients (227). The occasionally observed clinical resistance to azole drugs in some cases of seborrheic dermatitis could be attributed to variable genotypes of the recently described M. globosa azolemetabolizing CYP51 enzyme (177).…”

The Tetrazole VT-1161 Is a Potent Inhibitor of Trichophyton rubrum through Its Inhibition of T. rubrum CYP51

“…The antifungal agents most commonly used against T. rubrum are ketoconazole, fluconazole, terbinafine, and flucytosine (13). The prolonged treatment regimens often required have led to the emergence of azoleresistant T. rubrum strains, especially strains resistant to fluconazole (14)(15)(16)(17).…”