Phosphorylation of mouse glutamine-fructose-6-phosphate amidotransferase 2 (GFAT2) by cAMP-dependent protein kinase increases the enzyme activity

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 Km 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 cAMP-dependent 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.     

Purification and characterization of glutamine:fructose 6-phosphate amidotransferase from rat liver

The enzyme glutamine:fructose 6-phosphate amidotransferase (L-glutamine:D-fructose-6-phosphate amidotransferase; EC 2.6.1.16, GFAT) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate and glutamine. In view of the important role of GFAT in the hexosamine biosynthetic pathway, we have purified the enzyme from rat liver and characterized its physicochemical properties in comparison to those from the published microbial enzymes. The purified enzyme has a molecular mass of about 75 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. On a Sephacryl S-200 gel filtration column, the purified enzyme eluted in a single peak corresponding to a molecular mass of about 280 kDa, indicating that the active enzyme may be composed of four subunits. The N-terminal amino acid sequence of the purified enzyme was determined as X-G-I-F-A-Y-L-N-Y-H-X-P-R, where X indicates an unidentified residue. The K(M) values of the purified enzyme for fructose 6-phosphate and glutamine were 0.4 and 0.8 mM, respectively. The purified enzyme was inactivated by 4, 4'-dithiodipyridine, and the activity of the inactivated enzyme was restored by dithiothreitol. The inactivation followed pseudo first-order and saturation kinetics with the K(inact) of 5.0 microM. Kinetic studies also indicated that 4,4'-dithiodipyridine is a competitive inhibitor of the enzyme with respect to glutamine. Isolation and analysis of the cysteine-modified peptide indicated that Cys-1 was the modified site. Cys-1 has been suggested to play an important role in enzymatic activity of the Escherichia coli enzyme (M. N. Isupov, G. Obmolova, S. Butterworth, M. Badet-Denisot, B. Badet, I. Polikarpov, J. A. Littlechild, and A. Teplyakov, 1996, Structure 4, 801-810).     

Feedback inhibition of L-glutamine D-fructose 6-phosphate amidotransferase by uridine diphosphate N-acetylglucosamine in Neurospora crassa

The enzyme, l-glutamine d-fructose 6-phosphate amidotransferase (EC 2.6.1.16) of Neurospora crassa, which catalyzes the formation of glucosamine 6-phosphate was shown to be subject to feedback inhibition by uridine diphosphate N-acetyl-d-glucosamine (UDP-GlcNAc). The conclusion is based on the following observations. UDP-GlcNAc, the direct precursor of chitin, did not accumulate in the cell even when its utilization for the synthesis of cell wall chitin was interrupted by the antibiotic polyoxin D, a competitive inhibitor of the chitin synthetase (EC 2.4.1.16). Furthermore, the cellular level of UDP-GlcNAc rose in a short period of time when the amidotransferase was bypassed in vivo by the addition of glucosamine to the growing medium of the fungus. The amidotransferase was purified from N. crassa approximately 85-fold. Kinetic studies showed that UDP-GlcNAc was a potent and specific inhibitor of the amidotransferase, and that it did not alter the Michaelis constant for either l-glutamine or d-fructose 6-phosphate, suggesting that the inhibitor binds at a site on the enzyme distinct from the active site.     

A radiometric assay for glutamine:fructose-6-phosphate amidotransferase

Glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the first step in the biosynthesis of amino sugars by transferring the amino group from l-glutamine to the acceptor substrate, fructose 6-phosphate, generating the products glucosamine 6-phosphate and glutamic acid. We describe a method for the synthesis and purification of the substrate, fructose 6-phosphate, and methods for a radiometric assay of human GFAT1 that can be performed in either of two formats: a small disposable-column format and a high-throughput 96-well-plate format. The method performed in the column format can detect 1 pmol of glucosamine 6-phosphate, much less than that required by previously published assays that measure GlcN 6-phosphate. The column assay demonstrates a broad linear range with low variability. In both formats, the assay is linear with time and enzyme concentration and is highly reproducible. This method greatly improves the sensitivity and speed with which GFAT1 activity can be measured and facilitates direct kinetic measurement of the transferase activity.     

The mechanism of potent GTP cyclohydrolase I inhibition by 2,4-diamino-6-hydroxypyrimidine: requirement of the GTP cyclohydrolase I feedback regulatory protein

Inhibition of GTP cyclohydrolase I (GTPCH) has been used as a selective tool to assess the role of de novo synthesis of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) in a biological system. Toward this end, 2,4-diamino-6-hydroxypyrimidine (DAHP) has been used as the prototypical GTPCH inhibitor. Using a novel real-time kinetic microplate assay for GTPCH activity and purified prokaryote-expressed recombinant proteins, we show that potent inhibition by DAHP is not the result of a direct interaction with GTPCH. Rather, inhibition by DAHP in phosphate buffer occurs via an indirect mechanism that requires the presence of GTPCH feedback regulatory protein (GFRP). Notably, GFRP was previously discovered as the essential factor that reconstitutes inhibition of pure recombinant GTPCH by the pathway end product BH4. Thus, DAHP inhibits GTPCH by engaging the endogenous feedback inhibitory system. We further demonstrate that L-Phe fully reverses the inhibition of GTPCH by DAHP/GFRP, which is also a feature in common with inhibition by BH4/GFRP. These findings suggest that DAHP is not an indiscriminate inhibitor of GTPCH in biological systems; instead, it is predicted to preferentially attenuate GTPCH activity in cells that most abundantly express GFRP and/or contain the lowest levels of L-Phe.     

Bacteriolytic effect of cessation of glucosamine supply, induced by specific inhibition of glucosamine-6-phosphate synthetase

The antibiotic tetaine (bacilysin) and its C-terminal epoxyaminoacid--anticapsin--are powerful inhibitors of glucosamine-6-phosphate synthetase (EC 5.3.1.19.) in cell-free extracts of Escherichia coli K-12. Tetaine acts on growing cells as a bactericidal agent. This bactericidal action, measured from 10 to 160 muM concentration, is a consequence of the induction of lysis of growing cells. The induction of lysis by tetaine is compared with the lytic action of some beta-lactams. Hypertonic medium, destruction of the antibiotic, presence of chloramphenicol or the addition of N-acetylglucosamine protect E. coli K-12 cells against lysis induced by tetaine. These effects are compared with those observed in the presence of penicillin G. The results indicate that inhibition of early or late stages of peptidoglycan synthesis all result in more or less the same consequence, i.e. death via cell lysis.     

Kinetic properties of human placental glucose-6-phosphate dehydrogenase

The kinetic properties of placental glucose-6-phosphate dehydrogenase were studied, since this enzyme is expected to be an important component of the placental protection system. In this capacity it is also very important for the health of the fetus. The placental enzyme obeyed "Rapid Equilibrium Ordered Bi Bi" sequential kinetics with K(m) values of 40+/-8 microM for glucose-6-phosphate and 20+/-10 microM for NADP. Glucose-6-phosphate, 2-deoxyglucose-6-phosphate and galactose-6-phosphate were used with catalytic efficiencies (k(cat)/K(m)) of 7.4 x 10(6), 4.89 x 10(4) and 1.57 x 10(4) M(-1).s(-1), respectively. The K(m)app values for galactose-6-phosphate and for 2-deoxyglucose-6-phosphate were 10+/-2 and 0.87+/-0.06 mM. With galactose-6-phosphate as substrate, the same K(m) value for NADP as glucose-6-phosphate was obtained and it was independent of galactose-6-phosphate concentration. On the other hand, when 2-deoxyglucose-6-phosphate used as substrate, the K(m) for NADP decreased from 30+/-6 to 10+/-2 microM as the substrate concentration was increased from 0.3 to 1.5 mM. Deamino-NADP, but not NAD, was a coenzyme for placental glucose-6-phosphate dehydrogenase. The catalytic efficiencies of NADP and deamino-NADP (glucose-6-phosphate as substrate) were 1.48 x 10(7) and 4.80 x 10(6) M(-1)s(-1), respectively. With both coenzymes, a hyperbolic saturation and an inhibition above 300 microM coenzyme concentration, was observed. Human placental glucose-6-phosphate dehydrogenase was inhibited competitively by 2,3-diphosphoglycerate (K(i)=15+/-3 mM) and NADPH (K(i)=17.1+/-3.2 microM). The small dissociation constant for the G6PD:NADPH complex pointed to tight enzyme:NADPH binding and the important role of NADPH in the regulation of the pentose phosphate pathway.     

Identification of a novel serine phosphorylation site in human glutamine:fructose-6-phosphate amidotransferase isoform 1

Glutamine:fructose-6-phosphate amidotransferase (Gfat) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway. The increasing amount of evidence that links excess hexosamine biosynthesis with pathogenic complications of type II diabetes highlights the need to understand the regulation of Gfat. Previous studies showed that eukaryotic Gfat is subjected to feedback inhibition by UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) and to phosphorylation by cAMP-activated protein kinase A (PKA). In this study, overexpression of human Gfat isoform 1 (hGfat1) in insect cells revealed that hGfat1 is phosphorylated in vivo. Using matrix-assisted laser desorption/ionization and electrospray tandem mass spectrometry, we have identified Ser243 as a novel phosphorylation site. Biochemical properties of the wild type and the Ser243Glu mutant of hGfat1 overexpressed in Escherichia coli were compared. Our results provide evidence that phosphorylation at Ser243 stimulates glucosamine 6-phosphate-synthesizing activity, lowers amidohydrolyzing activity in the absence of fructose 6-phosphate (F6P) (glutaminase activity), and lowers Km(F6P) 2-fold, but has no effect on UDP-GlcNAc inhibition. On the basis of the sequence consensus, AMP-activated protein kinase and calcium/calmodulin-dependent kinase II were identified to phosphorylate specifically Ser243 in vitro. Phosphorylation by these two kinases results in an increase of enzymatic activity by 1.4-fold. These findings suggest for the first time that hGfat1 may be regulated by kinases other than PKA.     

The mechanism of sugar phosphate isomerization by glucosamine 6-phosphate synthase

Glucosamine 6-phosphate synthase converts fructose-6P into glucosamine-6P or glucose-6P depending on the presence or absence of glutamine. The isomerase activity is associated with a 40-kDa C-terminal domain, which has already been characterized crystallographically. Now the three-dimensional structures of the complexes with the reaction product glucose-6P and with the transition state analog 2-amino-2-deoxyglucitol-6P have been determined. Glucose-6P binds in a cyclic form whereas 2-amino-2-deoxyglucitol-6P is in an extended conformation. The information on ligand-protein interactions observed in the crystal structures together with the isotope exchange and site-directed mutagenesis data allow us to propose a mechanism of the isomerase activity of glucosamine-6P synthase. The sugar phosphate isomerization involves a ring opening step catalyzed by His504 and an enolization step with Glu488 catalyzing the hydrogen transfer from C1 to C2 of the substrate. The enediol intermediate is stabilized by a helix dipole and the epsilon-amino group of Lys603. Lys485 may play a role in deprotonating the hydroxyl O1 of the intermediate.     

Phosphorylation of human glutamine:fructose-6-phosphate amidotransferase by cAMP-dependent protein kinase at serine 205 blocks the enzyme activity

Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the rate-limiting enzyme in glucosamine synthesis. Prior studies from our laboratory indicated that activation of adenylate cyclase was associated with depletion of O-GlcNAc modification. This finding and evidence that human GFAT (hGFAT) might be regulated by cAMP-dependent protein kinase (PKA) led us to investigate the role of PKA in hGFAT function. We confirmed that adenylate cyclase activation by forskolin results in diminished O-GlcNAc modification of several cellular proteins which can be overcome by exposure of the cells to glucosamine but not glucose, suggesting the PKA activation results in depletion of UDP-GlcNAc for O-glycosylation. To determine if GFAT is indeed regulated by PKA, we expressed the active form of the enzyme using a vaccinia virus expression system and showed that the activity of the enzyme was to decrease to undetectable levels by PKA phosphorylation. We mapped the PKA phosphorylation sites with the aid of matrix-assisted laser desorption ionization mass spectroscopy and showed that the protein was stoichiometrically phosphorylated at serine 205 and also phosphorylated, to a lesser extent at serine 235. Mutagenesis studies indicated that the phosphorylation of serine 205 by PKA was necessary for the observed inhibition of enzyme activity while serine 235 phosphorylation played no observable role. The activity of GFAT is down-regulated by cAMP, thus placing regulation on the hexosamine pathway that is in concert with the energy requirements of the organism. During starvation, hormones acting through adenylate cyclase could direct the flux of glucose metabolism into energy production rather than into synthetic pathways that require hexosamines.     

An enzyme-coupled assay for amidotransferase activity of glucosamine-6-phosphate synthase

An assay for glucosamine-6-phosphate synthase using a yeast glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) as coupling enzyme was developed. GNA1 transfers the acetyl moiety from acetyl-coenzyme A (CoA) to glucosamine-6-phosphate, releasing coenzyme A. The assay measures the production of glucosamine-6-phosphate by either following the consumption of acetyl-CoA spectrophotometrically at 230nm or quantifying the free thiol with 5,5'-dithio-bis(2-nitrobenzoic acid) (Ellman's reagent) in a discontinuous manner. This method is simple to perform and can be adapted to a 96-well microtiter plate format, which will facilitate high-throughput inhibitor screening and mechanistic studies using purified GlmS.     

Molecular cloning, cDNA sequence, and bacterial expression of human glutamine:fructose-6-phosphate amidotransferase.

Glutamine:fructose-6-phosphate amidotransferase (GFAT) has recently been shown to be an insulin-regulated enzyme that plays a key role in the induction of insulin resistance in cultured cells. As a first step in understanding the molecular regulation of this enzyme the human form of this enzyme has been cloned and the functional protein has been expressed in Escherichia coli. A 3.1-kilobase cDNA was isolated which contains the complete coding region of 681 amino acids. Expression of the cDNA in E. coli produced a protein of approximately 77 kDa and increased GFAT activity 4.5-fold over endogenous bacterial levels. Recombinant GFAT activity was inhibited 51% by UDP-GlcNAc whereas bacterial GFAT activity was insensitive to inhibition by UDP-GlcNAc. On the basis of these results we conclude that: 1) functional human GFAT protein was expressed, and 2) the cloned human cDNA encodes both the catalytic and regulatory domains of GFAT since the recombinant GFAT was sensitive to UDP-GlcNAc. Overall, the development of cloned GFAT molecular probes should provide new insights into the development of insulin resistance by allowing quantitation of GFAT mRNA levels in pathophysiological states such as non-insulin-dependent diabetes mellitus and obesity.     

A simple and sensitive method for glutamine:fructose-6-phosphate amidotransferase assay

For measuring glutamine:fructose-6-phosphate amidotransferase (GFAT) activity in cultured cells, an enzyme method -GDH method- was set up with high-efficiency, high-sensitivity and simple operation by determining the formed glutamate. During the process of making samples, reduced glutathione (GSH, 5 mM) and glucose-6-phosphate Na2 (5 mM) were added to the buffer for scraping the cells. The range of protein content in the samples was 80-150 microg. In the GFAT activity assay, the end product reduced acetylpyridine adenine dinucleotide (APADH) was determined at 370 nm directly. The suitable concentrations of the reactants fructose-6-phosphate (F-6-P), glutamine, acetylpyridine adenine dinucleotide (APAD) and glutamate dehydrogenase (GDH) were 0.8, 6 and 0.3 mM and 6 U, respectively. However, the excess of APAD may interfere with the APADH measurement. The reaction time course was 90 min. The GFAT activity in 3T3-L1, L6, HepG2 and HIRc cells were 1.84-8.51 nmol glutamate/mg protein.min.     

Structural analysis of human glutamine:fructose-6-phosphate amidotransferase, a key regulator in type 2 diabetes

Glutamine:fructose-6-phosphate amidotransferase (GFAT) is a rate-limiting enzyme in the hexoamine biosynthetic pathway and plays an important role in type 2 diabetes. We now report the first structures of the isomerase domain of the human GFAT in the presence of cyclic glucose-6-phosphate and linear glucosamine-6-phosphate. The C-terminal tail including the active site displays a rigid conformation, similar to the corresponding Escherichia coli enzyme. The diversity of the CF helix near the active site suggests the helix is a major target for drug design. Our study provides insights into the development of therapeutic drugs for type 2 diabetes.     

Isolation and characterization of the GFA1 gene encoding the glutamine:fructose-6-phosphate amidotransferase of Candida albicans.

Glutamine:fructose-6-phosphate amidotransferase (glucosamine-6-phosphate synthase) catalyzes the first step of the hexosamine pathway required for the biosynthesis of cell wall precursors. The Candida albicans GFA1 gene was cloned by complementing a gfa1 mutation of Saccharomyces cerevisiae (previously known as gcn1-1; W. L. Whelan and C. E. Ballou, J. Bacteriol. 124:1545-1557, 1975). GFA1 encodes a predicted protein of 713 amino acids and is homologous to the corresponding gene from S. cerevisiae (72% identity at the nucleotide sequence level) as well as to the genes encoding glucosamine-6-phosphate synthases in bacteria and vertebrates. In cell extracts, the C. albicans enzyme was 4-fold more sensitive than the S. cerevisiae enzyme to UDP-N-acetylglucosamine (an inhibitor of the mammalian enzyme) and 2.5-fold more sensitive to N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (a glutamine analog and specific inhibitor of glucosamine-6-phosphate synthase). Cell extracts from the S. cerevisiae gfa1 strain transformed with the C. albicans GFA1 gene exhibited sensitivities to glucosamine-6-phosphate synthase inhibitors that were similar to those shown by the C. albicans enzyme. Southern hybridization indicated that a single GFA1 locus exists in the C. albicans genome. Quantitative Northern (RNA) analysis showed that the expression of GFA1 in C. albicans is regulated during growth: maximum mRNA levels were detected during early log phase. GFA1 mRNA levels increased following induction of the yeast-to-hyphal-form transition, but this was a response to fresh medium rather than to the morphological change.