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.     

Functional domains and interdomain communication in Candida albicans glucosamine-6-phosphate synthase

Functional and structural properties of several truncated or mutated variants of Candida albicans Gfa1p (glucosamine-6-phosphate synthase) were compared with those of the wild-type enzyme. Fragments encompassing residues 1-345 and 346-712 of Gfa1p, expressed heterogeneously in bacterial host as His6 fusions, were identified as the functional GAH (glutamine amidehydrolysing) and ISOM (hexose phosphate-isomerizing) domains respectively. It was found that the native GAH domain is monomeric, whereas the native ISOM domain forms tetramers, as does the whole enzyme. Spectrofluorimetric and kinetic studies of the isolated domains, the Delta218-283Gfa1p mutein and the wild-type enzyme revealed that the binding site for the feedback inhibitor, uridine 5'-diphospho-N-acetyl-D-glucosamine, is located in the ISOM domain. Inhibitor binding affects amidohydrolysing activity of the GAH domain and, as a consequence, the GlcN-6-P (D-glucosamine-6-phosphate)-synthetic activity of the whole enzyme. The fragment containing residues 218-283 is neither involved in ligand binding nor in protein oligomerization. Comparison of the catalytic activities of Gfa1p(V711F), Delta709-712Gfa1p, Gfa1p(W97F) and Gfa1p(W97G) with those of the native Gfa1p and the isolated domains provided evidence for an intramolecular channel connecting the GAH and ISOM domains of Gfa1p. The channel becomes leaky upon deletion of amino acids 709-712 and in the W97F and W97G mutants. The Trp97 residue was found to function as a molecular gate, opening and closing the channel. The W97G and V711F mutations resulted in an almost complete elimination of the GlcN-6-P-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities.     

Glucosamine-6-phosphate synthase from Escherichia coli yields two proteins upon limited proteolysis: identification of the glutamine amidohydrolase and 2R ketose/aldose isomerase-bearing domains based on their biochemical properties

The proteolysis of native glucosamine-6-phosphate synthase (Mr 67,000) from Escherichia coli was investigated using two nonspecific and five specific endoproteinases, alpha-chymotrypsin generated two nonoverlapping polypeptides CT1 and CT2 of Mr 40,000 and 27,000 lacking glucosamine-6P synthesizing activity. Amino terminal and carboxy terminal sequence analysis showed that cleavage occurred between positions 240 and 241 of the primary sequence without further degradation. The glutamine amidohydrolase activity was located in the CT2 N-terminal polypeptide which was capable of incorporating 0.7 equivalent of the glutamine site-directed affinity label [2-3H]-N3-(4-methoxyfumaroyl)-diaminopropionic acid indicating that it bears the amidotransferase function. CT1 which displayed a higher reactivity than CT2 for fructose-6P binding contains the ketose/aldose isomerase activity. These data suggest the existence of a hinge structure essential for the catalytically efficient coupling between the ammonia generating domain and the sugar binding domain and support the model recently proposed by Mei and Zalkin in which purF-type amidotransferases contain a glutamine hydrolase domain of approximately 200 amino acids fused to an ammonia-transfer domain.     

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.     

Direct inhibitory effect of gonadotropin releasing hormone in the uterus of rat

Estradiol induced increase in ornithine decarboxylase (ODC) and glucosamine-6-phosphate synthase activities of rat uterus were inhibited by simultaneous treatment with gonadotropin releasing hormone (GnRH) or its agonists. The direct inhibitory effect of GnRH analogs was found to be dose dependent. It was observed that a higher dose of GnRH analog was needed to cause inhibition of glucosamine-6-phosphate synthase when compared to ODC activity. The inhibitory effect of GnRH was not observed if its injection was delayed following estradiol treatment. These results show that the extra-pituitary inhibitory effects of GnRH involves enzymes associated with cell proliferation.     

Regulation by testosterone of the glucosamine-6-phosphate synthase activity in the male reproductive organs of rat

Accessory reproductive organs of male rat were found to contain high activities of glucosamine-6-phosphate synthase (glucosaminephosphate isomerase (glutamine-forming), EC 5.3.1.19). Castration caused drastic reduction (75%) in the enzyme activity of ventral prostate. Testosterone propionate administration restored the enzyme activity while cortisol and estradiol-17β did not cause any effect. Cycloheximide blocked the stimulation caused by testosterone.     

Cloning and sequence analysis of Histoplasma capsulatum glucosamine-6-phosphate synthase gene fragment

The 3' part of the glucosamine-6-phosphate synthase gene from Histoplasma capsulatum was PCR amplified using degenerate primers designed from the known glucosamine-6-phosphate synthase gene sequences, cloned and sequenced. The computer analysis of the 676 bp sequence revealed the presence of two introns. The identities of the deduced amino acid sequence to the corresponding Saccharomyces cerevisiae and Candida albicans fragment are 65 and 63.8%, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Directed evolution and characterization of Escherichia coli glucosamine synthase

Glucosamine synthase (GlmS) converts fructose-6-phosphate to glucosamine-6-phosphate. Overexpression of GlmS in Escherichia coli increased synthesis of glucosamine-6-P, which was dephosphorylated and secreted as glucosamine into the growth medium. The E. coli glmS gene was improved through error-prone polymerase chain reaction (PCR) in order to develop microbial strains for fermentation production of glucosamine. Mutants producing higher levels of glucosamine were identified by a plate cross-feeding assay and confirmed in shake flask cultures. Over 10 mutants were characterized and all showed significantly reduced sensitivity to inhibition by glucosamine-6-phosphate. Ki of mutants ranged from 1.4 to 4.0 mM as compared to 0.56 mM for the wild type enzyme. Product resistance resulted from single mutations (L468P, G471S) and/or combinations of mutations in the sugar isomerase domain. Most overexpressed GlmS protein was found in the form of inclusion bodies. Cell lysate from mutant 2123-72 contained twice as much soluble GlmS protein and enzyme activity as the strain overexpressing the wild type gene. Using the product-resistant mutant, glucosamine production was increased 60-fold.     

Isomerase activity of the C-terminal fructose-6-phosphate binding domain of glucosamine-6-phosphate synthase from Escherichia coli

The isomerase activity of the C-terminal fructose-6P binding domain (residues 241-608) of glucosamine-6-phosphate synthase from Escherichia coli has been studied. The equilibrium constant of the C-terminal domain k(eq) ([glucose-6P]/[fructose-6-P]) = 5.0. A non-competitive product inhibition of the isomerase activity by the reaction product glucose-6-P has been detected. The existence of more than one binding and reaction sites for the substrate fructose-6P on the molecule of glucosamine-6-phosphate synthase can be expected. The fructose-6P binding domain possibly includes a regulatory site, different from the catalytic center of the enzyme.     

Chemical modification of glucosamine-6-phosphate synthase by diethyl pyrocarbonate: evidence of histidine requirement for enzymatic activity.

Glucosamine-6-phosphate synthase from Escherichia coli was inactivated by diethylpyrocarbonate at pH 7.3 and 4 degrees C with a second-order rate constant of 1220 M-1 min-1. The difference spectrum of inactivated vs native enzyme had a maximum absorption at 242 nm, which is characteristic of N-carbethoxyhistidine. No trough at around 280 nm due to O-carbethoxytyrosine was observed and the sulfhydryl content of the enzyme was unchanged. Studies with [14C]diethylpyrocarbonate provided evidence that derivatization of a single histidine residue of the amino-terminal glutamine-binding domain inactivated glucosamine-6P synthase. These results are consistent with the participation of an histidine residue in a catalytic triad, Cys/His/Asp, necessary to generate ammonia from glutamine.     

Collective motions in glucosamine-6-phosphate synthase: influence of ligand binding and role in ammonia channelling and opening of the fructose-6-phosphate binding site

The large protein motions of the bacterial enzyme glucosamine-6-phosphate synthase have been addressed using full atom normal modes analysis for the empty, the glucose-6-phosphate and the glucose-6-phosphate + glutamate bound proteins. The approach that was used involving energy minimizations along the normal modes coordinates identified functional motions of the protein, some of which were characterized earlier by X-ray diffraction studies. This method made it possible for the first time to highlight significant energy differences according to whether none, only one or both of the active sites of the protein were occupied. Our data favoured a specific motion of the glutamine binding domain following the fixation of fructose-6-phosphate and suggested a rigidified structure with both sites occupied. Here, we show that most of the collective large amplitude motions of glucosamine-6-phosphate synthase that are modulated by ligand binding are crucial for the enzyme catalytic cycle, as they strongly modify the geometry of both the ammonia channel and the C-tail, demonstrating their role in ammonia transfer and ligand binding.     

Anticandidal properties of N-acylpeptides containing an inhibitor of glucosamine-6-phosphate synthase

A series of N-acyl peptides 1-9, containing an inhibitor of glucosamine-6-phosphate synthase have been synthesised and tested against Candida strains. N-Acylated peptides inhibit glucosamine-6-phosphate synthase in cell free extracts from Candida albicans. Antifungal activities of the tested compounds correlated with their lipophilic properties. Peptides acylated with decanoic acid were found to be the most potent in the series. N-decanoylpeptides also showed activity against Candida albicans Gu5 resistant mutant with Cdr1 and Cdr2 drug extrusion proteins that causes MDR by an active efflux mechanism.     

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.     

Copurification of glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase activities of Escherichia coli: characterization of the glmU gene product as a bifunctional enzyme catalyzing two subsequent steps in the pathway for UDP-N-acetylglucosamine synthesis.

The glmU gene product of Escherichia coli was recently identified as the N-acetylglucosamine-1-phosphate uridyltransferase activity which catalyzes the formation of UDP-N-acetylglucosamine, an essential precursor for cell wall peptidoglycan and lipopolysaccharide biosyntheses (D. Mengin-Lecreulx and J. van Heijenoort, J. Bacteriol. 175:6150-6157, 1993). Evidence that the purified GlmU protein is in fact a bifunctional enzyme which also catalyzes acetylation of glucosamine-1-phosphate, the preceding step in the same pathway, is now provided. Kinetic parameters of both reactions were investigated, indicating in particular that the acetyltransferase activity of the enzyme is fivefold higher than its uridyltransferase activity. In contrast to the uridyltransferase activity, which is quite stable and insensitive to thiol reagents, the acetyltransferase activity was rapidly lost when the enzyme was stored in the absence of reducing thiols or acetyl coenzyme A or was treated with thiol-alkylating agents, suggesting the presence of at least one essential cysteine residue in or near the active site. The acetyltransferase activity is greatly inhibited by its reaction product N-acetylglucosamine-1-phosphate and, interestingly, also by UDP-N-acetylmuramic acid, which is one of the first precursors specific for the peptidoglycan pathway. The detection in crude cell extracts of a phosphoglucosamine mutase activity finally confirms that the route from glucosamine-6-phosphate to UDP-N-acetylglucosamine occurs via glucosamine-1-phosphate in bacteria.     

Crystal structure of MJ1247 protein from M. jannaschii at 2.0 A resolution infers a molecular function of 3-hexulose-6-phosphate isomerase

The crystal structure of the hypothetical protein MJ1247 from Methanococccus jannaschii at 2 A resolution, a detailed sequence analysis, and biochemical assays infer its molecular function to be 3-hexulose-6-phosphate isomerase (PHI). In the dissimilatory ribulose monophosphate (RuMP) cycle, ribulose-5-phosphate is coupled to formaldehyde by the 3-hexulose-6-phosphate synthase (HPS), yielding hexulose-6-phosphate, which is then isomerized to fructose-6-phosphate by the enzyme 3-hexulose-6-phosphate isomerase. MJ1247 is an alpha/beta structure consisting of a five-stranded parallel beta sheet flanked on both sides by alpha helices, forming a three-layered alpha-beta-alpha sandwich. The fold represents the nucleotide binding motif of a flavodoxin type. MJ1247 is a tetramer in the crystal and in solution and each monomer has a folding similar to the isomerase domain of glucosamine-6-phosphate synthase (GlmS).     

Glucosamine-6-phosphate synthase--the multi-facets enzyme

L-Glutamine: D-fructose-6-phosphate amidotransferase, known under trivial name of glucosamine-6-phosphate synthase, as the only member of the amidotransferase subfamily of enzymes, does not display any ammonia-dependent activity. This enzyme, catalysing the first committed step in a pathway leading to the eventual formation of uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), is an important point of metabolic control in biosynthesis of amino sugar-containing macromolecules. The molecular mechanism of reaction catalysed by GlcN-6-P synthase is complex and involves both amino transfer and sugar isomerisation. Substantial alterations to the enzyme structure and properties have been detected in different neoplastic tissues. GlcN-6-P synthase is inflicted in phenomenon of hexosamine-induced insulin resistance in diabetes. Finally, this enzyme has been proposed as a promising target in antifungal chemotherapy. Most of these issues, especially their molecular aspects, have been extensively studied in recent years. This article provides a comprehensive overview of the present knowledge on this multi-facets enzyme.     

N3-haloacetyl derivatives of L-2,3-diaminopropanoic acid: novel inactivators of glucosamine-6-phosphate synthase.

N3-Haloacetyl derivatives of L-2,3-diaminopropanoic acid, novel glutamine analogs, were shown to be strong inhibitors of glucosamine-6-phosphate synthase from bacteria and Candida albicans. The inhibition was competitive with respect to glutamine and non-competitive with respect to D-fructose-6-phosphate. In the absence of glutamine, the tested compounds inactivated glucosamine-6-phosphate synthase from C. albicans with Kinact = 0.5 microM, 0.55 microM and 18.5 microM for bromoacetyl-, iodoacetyl- and chloroacetyl derivatives of L-2,3-diaminopropanoic acid, respectively. The inactivation obeyed the criteria for active site-directed modification.     

Purification to homogeneity of Candida albicans glucosamine-6-phosphate synthase overexpressed in Escherichia coli

The Candida albicans GFA1 gene encoding glucosamine-6-phosphate synthase, an enzyme of cell wall biosynthesis pathway in fungi and bacteria, recently an object of interest as a target for the chemotherapy of systemic mycoses, was PCR amplified and cloned to an Escherichia coli expression vector pET23b. The activity of the enzyme in the lysates from the overproducing E. coli strain was approximately 50-100 times higher than in the lysates from the control E. coli strain. This abundant overproduction allows to purify milligram amounts of the enzyme to homogeneity.     

Identification of the Pseudomonas aeruginosa glmM gene, encoding phosphoglucosamine mutase

A search for a potential algC homologue within the Pseudomonas aeruginosa PAO1 genome database has revealed an open reading frame (ORF) of unknown function, ORF540 in contig 54 (July 1999 Pseudomonas genome release), that theoretically coded for a 445-amino-acid-residue polypeptide (I. M. Tavares, J. H. Leit?o, A. M. Fialho, and I. Sá-Correia, Res. Microbiol. 150:105-116, 1999). The product of this gene is here identified as the phosphoglucosamine mutase (GlmM) which catalyzes the conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, an essential step in the formation of the cell wall precursor UDP-N-acetylglucosamine. The P. aeruginosa gene has been cloned into expression vectors and shown to restore normal peptidoglycan biosynthesis and cell growth of a glmM Escherichia coli mutant strain. The GlmM enzyme from P. aeruginosa has been overproduced to high levels and purified to homogeneity in a six-histidine-tagged form. Beside its phosphoglucosamine mutase activity, the P. aeruginosa enzyme is shown to exhibit phosphomannomutase and phosphoglucomutase activities, which represent about 20 and 2% of its GlmM activity, respectively.     

Studies on the mechanism of Escherichia coli glucosamine-6-phosphate isomerase.

Escherichia coli glucosamine-6-phosphate isomerase is specific for removal of the 1-pro-R hydrogen of fructose 6-phosphate (fructose-6-P). The conversion of [2-3H]glucosamine-6-P to fructose-6-P plus ammonia is accompanied by 99% exchange of tritium with water and 0.6% transfer to C-1 of fructose-6-P. The enzyme is active toward alpha-glucosamine-6-P and apparently inactive toward the beta anomer. The combination of the above results supports a cisenolamine intermediate for the reaction. The labeling of substrate and product pools in tritiated water shows that the two halves of the reaction are each freely reversible. No single step appears to be rate determining. 2-Amino-2-deoxyglucitol-6-P is an unusually strong competitive inhibitor (K1 = 2 X 10(-7) M, compared with the Km = 4 X 10(-4) M for glucosamine-6-P), suggesting the enzyme has a strong affinity for the open-chain form of glucosamine-6-P.