title: Heterologous expression and characterization of CYP61A1 from dandruff-causing Malassezia globosa.
abstract: Malassezia globosa is pathogenic fungus that causes skin disorders including dandruff in humans. Many yeast cytochrome CYP enzymes are involved in the biosynthesis of sterols and are considered major targets of azole antifungal agents. Here, we report on the expression and characterization of the MGL_0310 gene product (CYP61A1), a sterol C-22 desaturase in M. globosa. The open reading frame of the CYP61A1 gene was amplified by PCR from M. globosa CBS 7966 genomic DNA and cloned into a pCW vector. The CYP61A1 gene was heterologously expressed in Escherichia coli and purified using a Ni(2+)-NTA affinity column. The purified CYP61A1 protein exhibited a CO-difference spectrum typical of CYPs with a maximum absorption at 452nm. Binding spectral titration with β-sitosterol and campesterol demonstrated the type I binding mode with an increase at 411nm and a decrease at 432nm. The calculated Kd values are 5.4±0.6μM and 6.1±1.0μM for β-sitosterol and campesterol, respectively. No metabolic product, however, was observed in the CYP61A1-supported enzyme reaction with these sterols. The purified CYP61A1 protein exhibited tight binding to azole agents, suggesting that this enzyme may be a target for the pathogenic M. globosa fungus. Moreover, several fatty acids were found to bind to CYP61A1, indicating that the architecture of the enzyme includes a relatively large active site space. This study provides new insight into the biosynthesis of fungal sterols in M. globosa and a basis for the development of antifungal as potential therapeutic agents to treat dandruff.
authors.affiliation: College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea.
authors.forename: Seul-Ong
authors.initials: SO
authors.lastname: Ohk
authors.affiliation: Department of Biological Sciences, Konkuk University, Seoul 143-701, Republic of Korea.
authors.forename: Hyoung-Goo
authors.initials: HG
authors.lastname: Park
authors.affiliation: Department of Biological Sciences, Konkuk University, Seoul 143-701, Republic of Korea.
authors.forename: Hwayoun
authors.initials: H
authors.lastname: Lee
authors.affiliation: College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea.
authors.forename: Yeo-Jung
authors.initials: YJ
authors.lastname: Kwon
authors.affiliation: Department of Dermatology, College of Medicine, Chung-Ang University, Seoul 156-755, Republic of Korea.
authors.forename: Beom Joon
authors.initials: BJ
authors.lastname: Kim
authors.affiliation: Department of Biological Sciences, Konkuk University, Seoul 143-701, Republic of Korea. Electronic address: donghak@konkuk.ac.kr.
authors.forename: Donghak
authors.initials: D
authors.lastname: Kim
authors.affiliation: College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea. Electronic address: yjchun@cau.ac.kr.
authors.forename: Young-Jin
authors.initials: YJ
authors.lastname: Chun
mesh_terms: D000595:Amino Acid Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D063807:Dandruff / Q000382:microbiology*; D003881:Dermatomycoses / Q000382:microbiology; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008290:Malassezia / Q000235:genetics*; D008969:Molecular Sequence Data; D016415:Sequence Alignment
chemical_list: D005656:Fungal Proteins; D003577:Cytochrome P-450 Enzyme System
keywords: Azole; C22-desaturase; CYP; CYP61A1; Malassezia globosa; P450; Sterol