Control of inositol biosynthesis in Saccharomyces cerevisiae: properties of a repressible enzyme system in extracts of wild-type (Ino+) cells

Abstract

Inositol biosynthesis was studied in soluble, cell extracts of a wild-type (Ino+) strain of Saccharomyces cerevisiae. Two reactions were detected: (i) conversion of D-glucose-6-phosphate to a phosphorylated form of inositol, presumably inositol-l-phosphate (IP synthetase, EC 5.5.1.4), and (ii) conversion of phosphorylated inositol to inositol (IP phosphatase, EC 3.1.3.25). The in vitro rate of conversion of glucose-6-phosphate to inositol was proportional to incubation time and enzyme concentration. The pH optimum was 7.0. The synthesis of inositol required oxidized nicotinamide adenine dinucleotide (NAD+) and was stimulated by NH4Cl and MgCl2. NADP+ substituted poorly for NADI, and NADH inhibited the reaction. Phosphorylated inositol accumulated in the absence of MgCl2, suggesting that inositol-phosphate is an intermediate in the pathway and that Mg2+ ions stimulate the dephosphorylation of inositol-phosphate. IP synthetase was inhibited approximately 20% in the presence of inositol in the reaction mixture at concentrations exceeding 1 mM. The enzyme was repressed approximately 50-fold when inositol was present in the growth medium at concentrations exceeding 50 ,uM. IIP synthetase reached the fully repressed level approximately 10 h after the addition of inositol to logarithmic cultures grown in the absence of inositol. The specific activity ofthe enzyme increased with time in logarithmically growing cultures lacking inositol and approached the fully derepressed level as the cells entered stationary phase.

Regulation of inositol monophosphatase in Saccharomyces cerevisiae

“…Experiments to address this question await the cloning of the yeast monophosphatase gene(s), which is currently in progress in our laboratory. It is interesting to note that, while inositol represses expression of I-1-P synthase (Culbertson et al, 1976), which catalyses the first step in the de novo synthesis of inositol (conversion of glucose 6-P to I-1-P), inositol has the opposite effect on monophosphatase activity, which is required to dephosphorylate I-1-P to inositol. One possible explanation is that different monophosphatases carry out de novo inositol biosynthesis and synthesis of inositol from inositol polyphosphates derived from PIP 2 breakdown.…”

Inositol and higher inositol phosphates in neural tissues: homeostasis, metabolism and functional significance

“…Inositol is synthesized de novo from glucose in a number of organs, including brain, testis, kidney and liver (Hauser and Finelli 1963). The trimeric enzyme D-inositol-3-phosphate synthase catalyzes the irreversible cyclic aldol condensation of glucose-6-phosphate to yield D-inositol-3-phosphate (L-inositol-1-phosphate; Eisenberg 1967;Culbertson et al 1976;Maeda and Eisenberg 1980). The reaction requires nicotinamide adenine dinucleotide (oxidized) (NAD), is stimulated by NH 4 + and K + , and is inhibited by Li + .…”

Inositol biosynthesis: Candida albicans and Saccharomyces cerevisiae genes share common regulation

“…Inositol biosynthesis in S. cerevisiae has been previously examined. Biochemical studies revealed that the biosynthesis of inositol was repressed by exogenous inositol (Culbertson et al, 1976). Genetic studies indicated that there is a complex cascade of regulation.…”

“…The regulation of the I N 0 1 gene of S. cerevisiae is extremely complex. Inositol biosynthetic activity in S. cerevisiae is regulated by the presence or absence of inositol in the media (Culbertson et al, 1976). This regulation is reflected in the steady-state levels of the corresponding RNA (Hirsch and Henry, 1986) and in the amount of inositol-1-phosphate synthase found in the cells (Donahue and Henry, 1981).…”