Purification and Characterization of Two Isoenzymes of DL-Glycerol-3-phosphatase from Saccharomyces cerevisiae

Abstract

The existence of specific DL-glycerol-3-phosphatase (EC 3.1.3.21) activity in extracts of Saccharomyces cerevisiae was confirmed by examining strains lacking nonspecific acid and alkaline phosphatase activities. During purification of the glycerol-3-phosphatase, two isozymes having very similar molecular weights were isolated by gel filtration and anion exchange chromatography. By microsequencing of trypsin-generated peptides the corresponding genes were identified as previously sequenced open reading frames of unknown function. The two genes, GPP1 (YIL053W) and GPP2 (YER062C) encode proteins that show 95% amino acid identity and have molecular masses of 30.4 and 27.8 kDa, respectively. The intracellular concentration of Gpp2p increases in cells subjected to osmotic stress, while the production of Gpp1p is unaffected by changes of external osmolarity. Both isoforms have a high specificity for DL-glycerol-3-phosphate, pH optima at 6.5, and K m G3P in the range of 3-4 mM. The osmotic induction of Gpp2p is blocked in cells that are defective in the HOG-mitogenactivated protein kinase pathway, indicating that GPP2 is a target gene for this osmosensing signal transduction pathway. Together with DOG1 and DOG2, encoding two highly homologous enzymes that dephosphorylate 2-deoxyglucose-6-phosphate, GPP1 and GPP2 constitute a new family of genes for low molecular weight phosphatases.

Regulatory network of genes associated with stimuli sensing, signal transduction and physiological transformation of appressorium inMagnaporthe oryzae

“…Dihydroxyacetone, a glycolytic intermediate, is reduced to glycerol-3-phosphate by an NAD+-dependent glycerol phosphate dehydrogenase ( GPD ). Glycerol-3-phosphate is further dephosphorylated to glycerol by glycerol-3-phosphatase, which includes two homologs encoded by HOR 1 and HOR 2 in S. cerevisiae (Hirayama et al 1995, Norbeck et al 1996). Gene replacement of the two homologous genes namely GPD 1 and GPD 2 resulted in mutants with a decreased accumulation of intracellular glycerol and increased sensitivity to osmotic stress as compared to wild-type in S. cerevisiae (Albertyn et al 1994).…”

Yeast: World’s Finest Chef

“…Yet, one must keep in mind that with fermentation is associated culture growth and, biomass composition is more oxidized than glucose, consequently an excess of reducing equivalents may be attained. The way yeast circumvent this problem, under anaerobic conditions, consists on the production of glycerol by reduction of the glycolytic intermediate dihydroxyacetone phosphate to glycerol 3-phosphate catalysed by NAD + -dependent glycerol 3-phosphate dehydrogenase (encoded by the two isogenes GPD1 and GPD2), and its subsequent dephosphorylation due to the action of glycerol 3-phosphatase (encoded by GPP1 and GPP2) [14][15][16].…”

Expression of glycerol 3‐phosphate dehydrogenase gene (CvGPD1) in salt‐tolerant yeast Candida versatilis is stimulated by high concentrations of NaCl

“…This glycerol 3-phosphate is then dephosphorylated by glycerol phosphatase to yield glycerol (Thome and Trench, 1999;Thome, 2004Thome, , 2005. The glycerol 3-phosphatase was encoded by GPP1 and GPP2, and transcription of GPP1/2 is induced by osmotic stress (Norbeck et al, 1996;Pahlmann et al, 2001).…”