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Heterologous expression and characterization of CYP61A1 from dandruff-causing Malassezia globosa
Seul-Ong Ohk1, Hyoung-Goo Park2, Hwayoun Lee2
1College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea.
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.
Insights
Researchers characterized a key enzyme, CYP61A1, in the dandruff-causing fungus Malassezia globosa. This enzyme binds azole antifungals, suggesting it
Area of Science:
- Biochemistry
- Mycology
- Enzymology
Background:
- Malassezia globosa is a pathogenic fungus responsible for human skin conditions like dandruff.
- Cytochrome CYP enzymes in yeasts are crucial for sterol biosynthesis and are targets for azole antifungal drugs.
Purpose of the Study:
- To express and characterize the MGL_0310 gene product (CYP61A1), a sterol C-22 desaturase in M. globosa.
- To investigate the potential of CYP61A1 as a therapeutic target for antifungal drug development.
Main Methods:
- The CYP61A1 gene was amplified from M. globosa DNA and cloned into a pCW vector.
- Heterologous expression in Escherichia coli, followed by purification using Ni(2+)-NTA affinity chromatography.
- Characterization involved CO-difference spectroscopy and binding spectral titration with sterols and azole agents.
Main Results:
- Purified CYP61A1 showed a typical CYP CO-difference spectrum with a peak at 452nm.
- Binding studies indicated a Type I binding mode for beta-sitosterol and campesterol, with Kd values of 5.4±0.6μM and 6.1±1.0μM, respectively.
- CYP61A1 exhibited tight binding to azole agents and also bound several fatty acids, suggesting a large active site.
Conclusions:
- CYP61A1, a sterol C-22 desaturase in M. globosa, tightly binds azole antifungal agents.
- This enzyme represents a potential therapeutic target for developing new treatments against M. globosa infections, including dandruff.
- The enzyme's interaction with fatty acids suggests a broad active site, offering insights into fungal sterol biosynthesis.
