Identification of the substrate‐binding sites in the phosphatidylserine synthase from Candida albicans

Abstract

Systemic infections by Candida spp. have high mortality rates, and this is related, in part, to limitations in current antifungals. Thus, there is a need for novel antifungals. The phosphatidylserine (PS) synthase (annotated Cho1) from C. albicans has been identified as a potential drug target because [1] it is required for virulence in mouse models of Candidiasis, [2] it is absent in humans, and [3] it is highly conserved among fungal pathogens. Inhibitors of Cho1 could be used as lead compounds for drug development. Rational drug design is one way to develop specific inhibitors, but neither the substrate-enzyme interactions nor the substrate-binding sites have been well-defined for Cho1. Therefore, we aim to biochemically identify and characterize the substrate-binding sites of Cho1, which binds cytidine-diphosphate-diacylglycerol (CDP-DAG) and serine, and catalyzes their condensation to form PS. For the CDP-DAG-binding site, a conserved CDP-alcohol-binding (CAPT) motif is present in Cho1. We tested this site for its role in PS synthesis by mutating the conserved residues using standard alanine-scanning mutagenesis, and using two assays to measure the impact of these mutations on PS synthesis. The first assay measures the impact of PS synthesis in whole cells based on their growth. PS is used to synthesize phosphatidylethanolamine (PE), and in the absence of PS, C. albicans must acquire exogenous ethanolamine to make PE by a salvage pathway. Thus, mutants lacking PS synthase activity grow very poorly in media lacking ethanolamine. The second assay measures PS synthesis directly in crude membranes based on incorporation of [3H]serine into [3H]PS. These experiments revealed that, with the exception of one residue (R133), all alanine substitutions of the conserved amino acids within the fungal CAPT motif showed decreased Cho1 function. A subsequent R133E mutant displayed reduced activity compared to R133A, indicating residue R133 interacts with CDP-DAG in a dispensable manner. For the serine-binding site, by using computational tools, we predicted potential serine-binding residues. Using alanine-scanning mutagenesis, we found that some of these predicted residues are required for Cho1 function. Two of the alanine substitution mutants, L184A and R189A, were characterized regarding their Michaelis-Menten kinetics. The mutant L184A, which displayed enhanced activity both for growth and in vitro PS synthase activity, showed an increased Vmax and an unchanged Km. The gain-of-function mechanism of this mutation is currently unknown. On the contrary, R189A, which showed decreased growth and in vitro PS synthase activity, increased the Km for serine but not Vmax, indicating that the residue R189 is involved in serine-binding. We plan to continue to determine if other potential residues directly bind serine, as this could be useful in future rational inhibitor design studies.