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Title: Solubilization, purification, and characterization of the hexameric form of phosphatidylserine synthase from Candida albicans
Author(s): Zhou, Yue
Syed, Jawhar H.
Semchonok, Dmitry A.
Wright, Edward
Kyrilis, Fotis L.
Hamdi, Farzad
Kastritis, Panagiotis L.Look up in the Integrated Authority File of the German National Library
Bruce, Barry D.
Reynolds, Todd B.
Issue Date: 2023
Type: Article
Language: English
Abstract: Phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic candidiasis. Rational drug design or small molecule screening are effective ways to identify specific inhibitors of Cho1, but both will be facilitated by protein purification. Due to the transmembrane nature of Cho1, methods were needed to solubilize and purify the native form of Cho1. Here, we used six non-ionic detergents and three styrene maleic acids (SMAs) to solubilize an HA-tagged Cho1 protein from the total microsomal fractions. Blue native PAGE and immunoblot analysis revealed a single band corresponding to Cho1 in all detergent-solubilized fractions, while two bands were present in the SMA2000-solubilized fraction. Our enzymatic assay suggests that digitonin- or DDM-solubilized enzyme has the most PS synthase activity. Pull-downs of HA-tagged Cho1 from the digitonin-solubilized fraction reveal an apparent MW of Cho1 consistent with a hexamer. Furthermore, negative-staining electron microscopy analysis and AlphaFold2 structure prediction modeling suggest the hexamer is composed of a trimer of dimers. We purified Cho1 protein to near-homogeneity as a hexamer using affinity chromatography and TEV protease treatment, and optimized Cho1 enzyme activity for manganese and detergent concentrations, temperature (24 °C), and pH (8.0). The purified Cho1 has a Km for its substrate CDP-diacylglycerol of 72.20 μM with a Vmax of 0.079 nmol/(μg∗min) while exhibiting a sigmoidal kinetic curve for its other substrate serine, indicating cooperative binding. Purified hexameric Cho1 can potentially be used in downstream structure determination and small drug screening.
Open Access: Open access publication
License: (CC BY 4.0) Creative Commons Attribution 4.0(CC BY 4.0) Creative Commons Attribution 4.0
Journal Title: The journal of biological chemistry
Publisher: ASBMB Publications
Publisher Place: Bethesda, Md.
Volume: 299
Issue: 6
Original Publication: 10.1016/j.jbc.2023.104756
Page Start: 1
Page End: 16
Appears in Collections:Open Access Publikationen der MLU

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