Functional expression and characterization of CYP51 from dandruff-causing Malassezia globosa.
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.
Department of Biological Sciences, Konkuk University, Seoul, Korea College of Pharmacy, Chung-Ang University, Seoul, Korea.
Donghak
D
Kim
Young-Ran
YR
Lim
Seul Ong
SO
Ohk
Beom Joon
BJ
Kim
Young-Jin
YJ
Chun
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000935:Antifungal Agents / Q000378:metabolism; D001393:Azoles / Q000378:metabolism; D003001:Cloning, Molecular; D025141:Drug Resistance, Fungal; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008290:Malassezia / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013057:Spectrum Analysis; D058886:Sterol 14-Demethylase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014010:Tinea Versicolor / Q000382:microbiology
D000935:Antifungal Agents; D001393:Azoles; D005656:Fungal Proteins; D011994:Recombinant Proteins; D058886:Sterol 14-Demethylase