Mapping the Substrate-Binding Sites in the Phosphatidylserine Synthase in Candida albicans.

Yue Zhou,Todd B Reynolds,Chelsi D Cassilly

doi:10.3389/fcimb.2021.765266

Abstract

The fungal phosphatidylserine (PS) synthase, a membrane protein encoded by the CHO1 gene, is a potential drug target for pathogenic fungi, such as Candida albicans. However, both substrate-binding sites of C. albicans Cho1 have not been characterized. Cho1 has two substrates: cytidyldiphosphate-diacylglycerol (CDP-DAG) and serine. Previous studies identified a conserved CDP-alcohol phosphotransferase (CAPT) binding motif, which is present within Cho1. We tested the CAPT motif for its role in PS synthesis by mutating conserved residues using alanine substitution mutagenesis. PS synthase assays revealed that mutations in all but one conserved amino acid within the CAPT motif resulted in decreased Cho1 function. In contrast, there were no clear motifs in Cho1 for binding serine. Therefore, to identify the serine binding site, PS synthase sequences from three fungi were aligned with sequences of a similar enzyme, phosphatidylinositol (PI) synthase, from the same fungi. This revealed a motif that was unique to PS synthases. Using alanine substitution mutagenesis, we found that some of the residues in this motif are required for Cho1 function. Two alanine substitution mutants, L184A and R189A, exhibited contrasting impacts on PS synthase activity, and were characterized for their Michaelis-Menten kinetics. The L184A mutant displayed enhanced PS synthase activity and showed an increased V max. In contrast, R189A showed decreased PS synthase activity and increased K m for serine, suggesting that residue R189 is involved in serine binding. These results help to characterize PS synthase substrate binding, and should direct rational approaches for finding Cho1 inhibitors that may lead to better antifungals.

Highlights

Candida spp. are the most common causes of fungal infections in humans and are a major cause of fungi-associated mortality worldwide (Brown et al, 2012)
We found that only very high concentrations of these substrates could compete with L-serine, indicating that the enzyme seems to be specific for L-serine, which agrees with previous studies in S. cerevisiae and E. coli (Kanfer and Kennedy, 1964; Carson et al, 1982)
One exception lies in some Gram negative bacteria, such as E. coli, where this motif is not present within certain enzymes binding CDP-DAG (e.g., E. coli phosphatidylserine synthase, PssA), indicating divergence (Matsumoto, 1997)

Summary

Introduction

Candida spp. are the most common causes of fungal infections in humans and are a major cause of fungi-associated mortality worldwide (Brown et al, 2012) These species are versatile pathogens, with C. albicans being the most common. While they can infect almost all body sites, they are most prevalently seen in infections of the oral mucosa, vaginal mucosa, and bloodstream/deep organs (i.e., invasive mycoses) (Kullberg et al, 2002). Substrate-Binding Sites in Phosphatidylserine Synthase are only three classes of antifungals that are commonly used to treat systemic infections: echinocandins (e.g., caspofungin), azoles (e.g., itraconazole), and polyenes (e.g., amphotericin B) (Pfaller et al, 2012) These all have limitations, which include drug resistance for the echinocandins and azoles and patient toxicity for the polyene amphotericin B (Holeman Jr and Einstein, 1963; Ghannoum and Rice, 1999; Odds et al, 2003; Whaley et al, 2016). One classic approach is to identify virulence-related proteins within C. albicans that are not conserved in humans and exploit them as drug targets

Methods

Results

Conclusion