Properties of particulate and solubilized phosphatidylserine synthase activity from Saccharomyces cerevisiae. Inhibitory effect of choline in the growth medium.

Carson, Mary Anne; Atkinson, K D; Waechter, Charles J.

doi:10.1016/s0021-9258(18)34304-7

Cited by 55 publications

(12 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Furthermore, we probed the specificity of the active site of this protein for L-serine by competition assays with the closely related amino acids L-threonine and D-serine by an in vitro assay ( Cassilly et al., 2017 ). 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 ). To further reveal insights into the active sites in the present communication, we mapped and characterized residues that affect binding for both substrates in Cho1.…”

Section: Introductionsupporting

confidence: 92%

“…The PS synthase enzyme (originally denoted as: cytidine 5’-diphospho-1,2-diacyl-sn-glycerol: l -serine O-phosphatidyltransferase, gene name: CHO1 ) was first identified in Saccharomyces cerevisiae ( Atkinson et al., 1980a ; Atkinson et al., 1980b ; Kovac et al., 1980 ). Since then, characterization of S. cerevisiae Cho1 included protein solubilization and purification ( Carman and Matas, 1981 ; Bae-Lee and Carman, 1984 ), determination of Michaelis-Menton kinetics ( Carman and Matas, 1981 ; Carson et al., 1982 ; Bae-Lee and Carman, 1984 ), understanding regulation of Cho1 ( Carson et al., 1982 ; Poole et al., 1986 ; Bailis et al., 1987 ; Kelley et al., 1988 ; Kinney and Carman, 1988 ), and identifying the localization of the enzyme ( Kuchler et al., 1986 ; Kohlwein et al., 1988 ). The function of the first fungal pathogen Cho1 homolog was described in C. albicans and this protein was shown to be required for both systemic and oral Candida infection in the mouse model ( Chen et al., 2010 ; Davis et al., 2018 ).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Zhou

Cassilly

Reynolds

2021

Front. Cell. Infect. Microbiol.

View full text Add to dashboard Cite

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.

show abstract

Section: Introductionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

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

Zhou

Cassilly

Reynolds

2021

Front. Cell. Infect. Microbiol.

View full text Add to dashboard Cite

show abstract

“…If the reaction products PS and CMP are present at higher concentrations, MjPSS can also catalyze the reverse reaction (Supplementary Fig. 1e ), similar to PSS of Saccharomyces cerevisiae 39 . However, the reverse reaction is not catalyzed when PA is used instead of PS, indicating that the binding of PS by MjPSS is also highly specific.…”

Section: Resultsmentioning

confidence: 95%

Crystal structures of phosphatidyl serine synthase PSS reveal the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases

et al. 2021

View full text Add to dashboard Cite

Phospholipids are the major components of the membrane in all type of cells and organelles. They also are critical for cell metabolism, signal transduction, the immune system and other critical cell functions. The biosynthesis of phospholipids is a complex multi-step process with high-energy intermediates. Several enzymes in different metabolic pathways are involved in the initial phospholipid synthesis and its subsequent conversion. While the “Kennedy pathway” is the main pathway in mammalian cells, in bacteria and lower eukaryotes the precursor CDP-DAG is used in the de novo pathway by CDP-DAG alcohol O-phosphatidyl transferases to synthetize the basic lipids. Here we present the high-resolution structures of phosphatidyl serine synthase from Methanocaldococcus jannaschii crystallized in four different states. Detailed structural and functional analysis of the different structures allowed us to identify the substrate binding site and show how CDP-DAG, serine and two essential metal ions are bound and oriented relative to each other. In close proximity to the substrate binding site, two anions were identified that appear to be highly important for the reaction. The structural findings were confirmed by functional activity assays and suggest a model for the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases, which synthetize the phospholipids essential for the cells.

show abstract

“…The utilization of the DGdependent pathway for PC synthesis is primarily used by wild-type cells grown in the presence of choline (Carman & Henry, 1989;Carman, 1989;Paltauf et al, 1992). This pathway becomes more important for PC synthesis when the enzymes in the CDP-DG-dependent pathway (Figure 1) are repressed (Homann et al, 1985;Klig et al, 1985Klig et al, , 1988Poole et al, 1986;Bailis et al, 1987;Carson et al, 1982;Overmeyer & Waechter, 1991;Lamping et al, 1991;Gaynor et al, 1991;Yamashita et al, 1982;Yamashita & Oshima, 1980;Waechter & Lester, 1973) or defective (Atkinson et al, 1980a,b;Kodaki & Yamashita, 1987, 1989Summers et al, 1988;McGraw & Henry, 1989). PA phosphatase also catalyzes the committed step in the synthesis of triacylglycerols (Figure 1).…”

mentioning

confidence: 99%

Regulation of Phosphatidate Phosphatase Activity from the YeastSaccharomyces cerevisiaeby Phospholipids

Carman

1996

Biochemistry

View full text Add to dashboard Cite

Regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by phospholipids was examined using purified enzyme and Triton X-100/phospholipid-mixed micelles. Anionic phospholipids activated phosphatidate phosphatase activity whereas zwitterionic phospholipids had a slight inhibitory effect on activity. Cardiolipin (A0.5 = 1.9 mol %), CDP-diacylglycerol (A0.5 = 2.6 mol %), and phosphatidylinositol (A0.5 = 5.5 mol %) were the most potent anionic phospholipid activators. Enzyme activation by cardiolipin (n=2.8), CDP-diacylglycerol (n=2.1), and phosphatidylinositol (n=3.3) followed positive cooperative kinetics. A kinetic analysis was performed to determine the mechanism of phosphatidate phosphatase activation by anionic phospholipids. The dependence of phosphatidate phosphatase on phosphatidate was cooperative (n approximately 2.2) in the absence and presence of phospholipid activators. Cardiolipin, CDP-diacylglycerol, and phosphatidylinositol were mixed competitive activators of phosphatidate phosphatase activity. The major effect of the activators was to cause a decrease in the Km for phosphatidate. Sphinganine, a positively charged sphingoid base, inhibited phosphatidate phosphatase activity and antagonized the activation of the enzyme by cardiolipin and phosphatidylinositol. Sphinganine caused an increase in the cooperativity of cardiolipin activation, but had little effect on the A0.5 value for cardiolipin. On the other hand, sphinganine had little effect on the cooperativity of phosphatidylinositol activation, but caused an increase in the A0.5 value for phosphatidylinositol. The activation constants for cardiolipin, CDP-diacylglycerol, and phosphatidylinositol were within the range of their cellular concentrations. These results suggested that the activation of phosphatidate phosphatase activity by anionic phospholipids may be physiologically relevant.

show abstract

Properties of particulate and solubilized phosphatidylserine synthase activity from Saccharomyces cerevisiae. Inhibitory effect of choline in the growth medium.

Cited by 55 publications

References 34 publications

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

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

Crystal structures of phosphatidyl serine synthase PSS reveal the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases

Regulation of Phosphatidate Phosphatase Activity from the YeastSaccharomyces cerevisiaeby Phospholipids

Contact Info

Product

Resources

About