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

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 Vmax. In contrast, R189A showed decreased PS synthase activity and increased Km 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.

Cellular Labeling of Phosphatidylserine Using Clickable Serine Probes

“…As an initial step to directly evaluate whether these serine probes interact with PS biosynthetic machinery, we assessed their ability to inhibit the activity of PS synthase (Cho1p) from C. albicans for the conversion of natural serine to PS. These experiments were performed using C. albicans cells because this organism is similar to S. cerevisiae in terms of the PS biosynthetic pathway and its PS synthase enzyme (alignment of Ca Cho1 and Sc Cho1 amino acid sequences in Clustal Omega revealed 60.52% sequence identity), and because we have previously developed a robust assay utilizing cell membrane extracts containing Cho1p to convert tritium-labeled serine ( 3 H-serine) and CDP-DAG into isotopically labeled PS ( 3 H-PS). ,, These experiments showed that 3 H-PS production was diminished by probes N- l -SerN 3 and C- l -SerN 3 in a dose-dependent manner (Figure ). Statistical analysis showed that both N- l -SerN 3 and C- l -SerN 3 displayed significantly decreased enzyme activity for all concentrations tested (1, 5, 10, and 25 mM).…”

The small molecule CBR-5884 inhibits theCandida albicansphosphatidylserine synthase

“…To further validate the inhibitory effects of the six molecules on Cho1, a radioactive PS synthase assay was conducted on Cho1 in the presence of these compounds. Unlike the malachite green-based assay, the radioactive PS synthase assay directly measures the incorporation of L-[ 3 H]-serine into PS in the lipid phase (26, 33, 47). The radioactive PS synthase assay was performed on purified Cho1 in the presence of the six compounds at 100 μM, and all six molecules were shown to totally inhibit Cho1 (Figure 3A), consistent with the screening results.…”

“…PS is the lipid precursor for making phosphatidylethanolamine (PE), an essential phospholipid, via the de novo pathway. Growth can resume when PS synthesis is inhibited, albeit more slowly, if the organism is able to make PE from ethanolamine acquired from the medium via the salvage (Kennedy) pathway (14, 26). Thus, ethanolamine-dependent growth in minimal media is a characteristic phenotype of PS synthesis loss.…”

“…Additionally, valuable insights have been provided into the serine-binding pocket from the atomic structure of the PS synthase from the archaean Methanocaldococcus jannaschii (32). Differences between the M. jannaschii PS synthase and C. albicans Cho1 are apparent by the presence of specific residues in C. albicans Cho1 that play important roles, yet lack clear counterparts in the M. jannaschii PS synthase (26, 32).…”

“…Cho1 activity has been measured in crude membrane preps (26, 31, 45, 47) and as a purified form (33, 48), using a radioactive substrate. However, that methodology is inconsistent with a high throughput screen.…”