Oligomeric Structure and Regulation of Candida albicans Glucosamine-6-phosphate Synthase

Milewski, Sławomir; Kuszczak, Danuta; Jedrzejczak, R.; Smith, Rachel J.; Brown, Alistair J. P.; Gooday, Graham W.

doi:10.1074/jbc.274.7.4000

Cited by 54 publications

(62 citation statements)

References 48 publications

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“…Although the GFAT enzymes used in these experiments were recombinant and tagged, studies of the enzyme purified from rat liver (17), Candida albicans (30), and Drosophila (31) all show stimulation of GFAT activity by PKA. Because the two mammalian GFATs are homologous and similar in size, it remains possible that GFAT2 was purified, thereby accounting for the stimulation by PKA that was identical to that which we observed for GFAT2.…”

Section: Discussionmentioning

confidence: 99%

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Phosphorylation of Mouse Glutamine-Fructose-6-phosphate Amidotransferase 2 (GFAT2) by cAMP-dependent Protein Kinase Increases the Enzyme Activity

Hu¹,

Riesland²,

Paterson

et al. 2004

Journal of Biological Chemistry

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A protein encoded by a new gene with approximately 75% homology to glutamine-fructose-6-phosphate amidotransferase (GFAT) was termed GFAT2 on the basis of this similarity. The mouse GFAT2 cDNA was cloned, and the protein was expressed with either an N-terminal glutathione S-transferase or His tag. The purified protein expressed in mammalian cells had GFAT activity. The K m values for the two substrates of reaction, fructose 6-phosphate and glutamine, were determined to be 0.8 mM for fructose 6-phosphate and 1.2 mM for glutamine, which are within the ranges determined for GFAT1. The protein sequence around the serine 202 of GFAT2 was conserved to the serine 205 of GFAT1, whereas the serine at 235 in GFAT1 was not present in GFAT2. Previously we showed that phosphorylation of serine 205 in GFAT1 by the catalytic subunit of cAMPdependent protein kinase (PKA) inhibits its activity. Like GFAT1, GFAT2 was phosphorylated by PKA, but GFAT2 activity increased approximately 2.2-fold by this modification. When serine 202 of GFAT2 was mutated to an alanine, the enzyme not only became resistant to phosphorylation, but also the increase in activity in response to PKA also was blocked. These results indicated that the phosphorylation of serine 202 was necessary and sufficient for these alterations by PKA. GFAT2 was modestly inhibited (15%) by UDP-GlcNAc but not through detectable O-glycosylation. GFAT2 is, therefore, an isoenzyme of GFAT1, but its regulation by cAMP is the opposite, allowing differential regulation of the hexosamine pathway in specialized tissues.

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Section: Discussionmentioning

confidence: 99%

“…The wild type and mutant samples contained the same amount of GST fusion protein, although the control sample contained no fusion protein when analyzed by Western blot (WB) using anti-GST antibody (Ab) (lower panel). (30). GST-mGFAT2, after treatment with phosphatase, was either untreated or treated with GST-OGT.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of Mouse Glutamine-Fructose-6-phosphate Amidotransferase 2 (GFAT2) by cAMP-dependent Protein Kinase Increases the Enzyme Activity

Hu¹,

Riesland²,

Paterson

et al. 2004

Journal of Biological Chemistry

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“…Unlike other amidotransferases in this class, neither bacterial nor mammalian forms of GFAT are capable of utilizing NH 3 as a nitrogen donor (7-9). Huynh et al (10) reported the characterization of rat liver GFAT, and Milewski et al (8) reported the biochemical properties of the Candida enzyme, which exhibits similarities to the mammalian forms. Recent descriptions of the GFAT1 isoform, GFAT1Alt, demonstrated that GFAT1 and GFAT1Alt differ in their sensitivity to inhibition by UDP-GlcNAc (11,12).…”

mentioning

confidence: 99%

Kinetic Characterization of Human Glutamine-fructose-6-phosphate Amidotransferase I

Broschat¹,

Gorka²,

Page³

et al. 2002

Journal of Biological Chemistry

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Glutamine-fructose-6-phosphate amidotransferase (GFAT) catalyzes the first committed step in the pathway for biosynthesis of hexosamines in mammals. A member of the N-terminal nucleophile class of amidotransferases, GFAT transfers the amino group from the L-glutamine amide to D-fructose 6-phosphate, producing glutamic acid and glucosamine 6-phosphate. The kinetic constants reported previously for mammalian GFAT implicate a relatively low affinity for the acceptor substrate, fructose 6-phosphate (Fru-6-P, K m 0.2-1 mM). Utilizing a new sensitive assay that measures the production of glucosamine 6-phosphate (GlcN-6-P), purified recombinant human GFAT1 (hGFAT1) exhibited a K m for Fru-6-P of 7 M, and was highly sensitive to product inhibition by GlcN-6-P. In a second assay method that measures the stimulation of glutaminase activity, a K d of 2 M was measured for Fru-6-P binding to hGFAT1. Further, we report that the product, GlcN-6-P, is a potent competitive inhibitor for the Fru-6-P site, with a K i measured of 6 M. Unlike other members of the amidotransferase family, where glutamate production is loosely coupled to amide transfer, we have demonstrated that hGFAT1 production of glutamate and GlcN-6-P are strictly coupled in the absence of inhibitors. Similar to other amidotransferases, competitive inhibitors that bind at the synthase site may inhibit the synthase activity without inhibiting the glutaminase activity at the hydrolase domain. GlcN-6-P, for example, inhibited the transfer reaction while fully activating the glutaminase activity at the hydrolase domain. Inhibition of hGFAT1 by the end product of the pathway, UDPGlcNAc, was competitive with a K i of 4 M. These data suggest that hGFAT1 is fully active at physiological levels of Fru-6-P and may be regulated by its product GlcN-6-P in addition to the pathway end product, UDP-GlcNAc.

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“…Gfa1p, the first enzyme in the pathway, is a key step in UDPGlcNAc biosynthesis; it is regulated at the transcriptional and posttranscriptional levels. Its activity increases in the yeast Candida albicans during hyphal growth (36) and in S. cerevisiae during mating, which correlates with an increase in chitin formation (54). The enzyme is inhibited by UDP-GlcNAc in C. albicans (36), Drosophila melanogaster (20), and bacteria (26).…”

mentioning

confidence: 99%

“…Its activity increases in the yeast Candida albicans during hyphal growth (36) and in S. cerevisiae during mating, which correlates with an increase in chitin formation (54). The enzyme is inhibited by UDP-GlcNAc in C. albicans (36), Drosophila melanogaster (20), and bacteria (26). Gfa1p activity is regulated by a protein kinase(s); the protein kinase A-dependent phosphorylated form of Gfa1p appears to have a higher activity than the unphosphorylated protein (20,57).…”

mentioning

confidence: 99%

Chitin Synthesis in Saccharomyces cerevisiae in Response to Supplementation of Growth Medium withGlucosamine and Cell WallStress

et al. 2003

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In Saccharomyces cerevisiae most chitin is synthesized by Chs3p, which deposits chitin in the lateral cell wall and in the bud-neck region during cell division. We have recently found that addition of glucosamine (GlcN) to the growth medium leads to a three-to fourfold increase in cell wall chitin levels. We compared this result to the increases in cellular chitin levels associated with cell wall stress and with treatment of yeast with mating pheromone. Since all three phenomena lead to increases in precursors of chitin, we hypothesized that chitin synthesis is at least in part directly regulated by the size of this pool. This hypothesis was strengthened by our finding that addition of GlcN to the growth medium causes a rapid increase in chitin synthesis without any pronounced change in the expression of more than 6,000 genes monitored with Affymetrix gene expression chips. In other studies we found that the specific activity of Chs3p is higher in the total membrane fractions from cells grown in GlcN and from mutants with weakened cell walls. Sucrose gradient analysis shows that Chs3p is present in an inactive form in what may be Golgi compartments but as an active enzyme in other intracellular membrane-bound vesicles, as well as in the plasma membrane. We conclude that Chs3p-dependent chitin synthesis in S. cerevisiae is regulated both by the levels of intermediates of the UDP-GlcNAc biosynthetic pathway and by an increase in the activity of the enzyme in the plasma membrane.

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Oligomeric Structure and Regulation of Candida albicans Glucosamine-6-phosphate Synthase

Cited by 54 publications

References 48 publications

Phosphorylation of Mouse Glutamine-Fructose-6-phosphate Amidotransferase 2 (GFAT2) by cAMP-dependent Protein Kinase Increases the Enzyme Activity

Phosphorylation of Mouse Glutamine-Fructose-6-phosphate Amidotransferase 2 (GFAT2) by cAMP-dependent Protein Kinase Increases the Enzyme Activity

Kinetic Characterization of Human Glutamine-fructose-6-phosphate Amidotransferase I

Chitin Synthesis in Saccharomyces cerevisiae in Response to Supplementation of Growth Medium withGlucosamine and Cell WallStress

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