Glucosamine 6-phosphate N-acetyltransferase Research Articles

Knockdown of the glucosamine-6-phosphate N-acetyltransferase gene by RNA interference enhances the virulence of entomopathogenic fungi against rice leaffolder Cnaphalocrocis medinalis

Insect cuticle acts as a first line of defense and a physical protective barrier against entomopathogens. Chitin biosynthesis pathway plays a crucial role in chitin formation in the cuticle of insects. Glucosamine-6-phosphate N-acetyltransferase (GNA) is a key enzyme in insect chitin biosynthesis that regulate the chitin formation. However, how GNA-mediated cuticle metabolism influences virulence of entomopathogenic fungi is still unknown. In this study, CmGNA gene was cloned and characterized from the rice leaffolder Cnaphalocrocis medinalis. The CmGNA contains an open read frame (ORF) 600 nucleotides, encoding 199 amino acids with an isoelectric point of 8.65 and a molecular weight of 22.30 kDa. The expression profile showed that CmGNA was highly expressed in 4th instar larvae and in the cuticle. Here, we also reported the impact of CmGNA gene and entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana, on expression pattern of chitin biosynthesis genes, feeding behavior, survival rate and average body weight of infected larvae, phenotypic deformities, rate of pupation, and adult emergence. Our results showed that knockdown of CmGNA and application of M. anisopliae and B. bassiana three days after RNA interference (RNAi) significantly decreased the expression of CmGNA and other associated genes, reduced feeding efficiency and survival rate, and caused loss of average body weight, less rate of pupation and adult emergence of infected larvae. Knockdown of CmGNA gene also increased the lethality of larvae caused by M. anisopliae and B. bassiana and resulted in significantly phenotypic deformities of infected larvae. Our findings illustrated that RNAi-mediated CmGNA knockdown disturbed the chitin synthesis genes that led to enhancing the virulence of M. anisopliae and B. bassiana, which can provide us new insights to develop novel biocontrol strategies against C. medinalis.

GNPNAT1 Serves as a Prognostic Biomarker Correlated with Immune Infiltration and Promotes Cancer Cell Metastasis through Stabilization of Snai2 in Lung Adenocarcinoma.

Lung cancer is a common malignant tumor with high morbidity and mortality rate. Glucosamine 6-phosphate N-acetyltransferase (GNPNAT1), which serves as a critical enzyme in hexosamine biosynthetic pathway (HBP), has been identified as a metastasis-associated gene and is upregulated in lung adenocarcinoma (LUAD). However, the exact role and related mechanism of GNPNAT1 in LUAD metastasis remain unknown. We analyzed the expression of GNPNAT1 in the public databases and confirmed the results by immunohistochemistry (IHC). The biological functions of GNPNAT1 in LUAD were investigated based on The Cancer Genome Atlas (TCGA). Correlations between GNPNAT1 and cancer immune characteristics were analyzed via the Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) and Cell-type Identification by Estimating Relative Subsets of RNA Transcript (CIBERSORT) R package. The underlying mechanisms of altered GNPNAT1 expression on LUAD cell tumorigenesis, proliferation, migration, invasion, and metastasis were explored in vitro and in vivo. We demonstrated that GNPNAT1 expression was significantly increased in LUAD and negatively associated with the overall survival (OS) of patients. hsa-miR-1-3p and hsa-miR-26a-5p were identified as upstream miRNA targets of GNPNAT1. GNPNAT1 was associated with the infiltration levels of CD8 T cells, memory-activated CD4 T cells, NK cells resting, macrophages M0, macrophages M1, neutrophils, gamma delta T cells, and eosinophils, while it was negatively correlated with memory-resting CD4 T cells, regulatory T cells (Tregs), resting NK cells, monocytes, resting dendritic cells, and resting mast cells. GNPNAT1 knockdown significantly inhibited proliferation, migration, invasion, epithelial-mesenchymal transition (EMT) process, and metastasis of LUAD cells, while overexpression of GNPNAT1 revealed the opposite effects. Rescue assay showed that Snai2 knockdown reversed GNPNAT1-induced LUAD cells migration, invasion, and EMT. Mechanistically, GNPNAT1 promoted cancer cell metastasis via repressing ubiquitination degradation of Snai2 in LUAD. Taken together, these data indicate that GNPNAT1 serves as a prognostic biomarker for LUAD patient. Additionally, GNPNAT1 is critical for promoting tumorigenesis and metastasis of LUAD cells and may be a potential therapeutic target for preventing LUAD metastasis.

The Transcription Factor CcRlm1 Regulates Cell Wall Maintenance and Poplar Defense Response by Directly Targeting CcChs6 and CcGna1 in Cytospora chrysosperma.

Maintenance of cell wall integrity is important for fungal cell morphology against external stresses and even virulence. Although the transcription factor Rlm1 is known to play major regulatory roles in the maintenance of cell integrity, the underlying mechanism of how Rlm1 contributes to cell wall integrity and virulence in phytopathogenic fungi remains unclear. Here, we demonstrated that CcRlm1 plays important roles in cell wall maintenance and virulence in the poplar canker fungus Cytospora chrysosperma. Among putative downstream targets, CcChs6 (chitin synthase) and CcGna1 (glucosamine 6-phosphate N-acetyltransferase) were found to be direct targets of CcRlm1 and shown to function in chitin synthesis and virulence. Furthermore, we found stronger induction of poplar defense responses when challenged with these gene deletion mutants. Collectively, these results suggest that CcRlm1 plays a critical role in the regulation of cell wall maintenance, stress response, and virulence by directly regulating CcChs6 and CcGna1 in C. chrysosperma. IMPORTANCE Cytospora chrysosperma causes canker diseases on woody plants, and the molecular basis of its infection is not well understood. This study shows that CcRlm1 is the major regulator of chitin synthesis and virulence of the poplar canker fungus. Our research contributes to further understanding the molecular basis of the interaction between C. chrysosperma and poplar.

GNPNAT1 is a potential biomarker correlated with immune infiltration and immunotherapy outcome in breast cancer.

Glucosamine 6-phosphate N-acetyltransferase (GNPNAT1) is a crucial enzyme involving hexosamine biosynthesis pathway and is upregulated in breast cancer (BRCA). However, its biological function and mechanism on patients in BRCA have not been investigated. In this study, the differential expression of GNPNAT1 was analyzed between BRCA tissues and normal breast tissues using the Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database, which was validated by quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry. Then, the potential clinical value of GNPNAT1 in BRCA was investigated based on TCGA database. Functional enrichment analyses, including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Gene Set Variation Analysis, were performed to explore the potential signaling pathways and biological functions involved in GNPNAT1 in BRCA. Tumor immune infiltration was analyzed using ESTIMATE, CIBERSORT and TISIDB database; and immune therapy response scores were assessed using TIDE. Finally, Western blot, Cell counting kit-8 and Transwell assay were used to determine the proliferation and invasion abilities of breast cancer cells with GNPNAT1 knockdown. GNPNAT1 was up-regulated in BRCA tissues compared with normal tissues which was subsequently verified in different cell lines and clinical tissue samples. Based on TCGA and GEO, the overexpression of GNPNAT1 in BRCA contributed to a significant decline in overall survive and disease specific survive. Functional enrichment analyses indicated that the enriched pathways in high GNPNAT1 expression group included citrate cycle, N-glycan biosynthesis, DNA repair, and basal transcription factors. Moreover, the overexpression of GNPNAT1 was negatively correlated with immunotherapy response and the levels of immune cell infiltration of CD8+ T cells, B cells, natural killer cells, dendritic cells and macrophages. Knockdown of GNPNAT1 impairs the proliferation and invasion abilities of breast cancer cells. GNPNAT1 is a potential diagnostic, prognostic biomarker and novel target for intervention in BRCA.

Novel form of rhizomelic skeletal dysplasia associated with a homozygous variant in GNPNAT1

BackgroundStudies exploring molecular mechanisms underlying congenital skeletal disorders have revealed novel regulators of skeletal homeostasis and shown protein glycosylation to play an important role.ObjectiveTo identify the genetic cause of rhizomelic...

Targeting a critical step in fungal hexosamine biosynthesis

Aspergillus fumigatus is a human opportunistic fungal pathogen whose cell wall protects it from the extracellular environment including host defenses. Chitin, an essential component of the fungal cell wall, is synthesized from UDP-GlcNAc produced in the hexosamine biosynthetic pathway. As this pathway is critical for fungal cell wall integrity, the hexosamine biosynthesis enzymes represent potential targets of antifungal drugs. Here, we provide genetic and chemical evidence that glucosamine 6-phosphate N-acetyltransferase (Gna1), a key enzyme in this pathway, is an exploitable antifungal drug target. GNA1 deletion resulted in loss of fungal viability and disruption of the cell wall, phenotypes that could be rescued by exogenous GlcNAc, the product of the Gna1 enzyme. In a murine model of aspergillosis, the Δgna1 mutant strain exhibited attenuated virulence. Using a fragment-based approach, we discovered a small heterocyclic scaffold that binds proximal to the Gna1 active site and can be optimized to a selective submicromolar binder. Taken together, we have provided genetic, structural, and chemical evidence that Gna1 is an antifungal target in A. fumigatus.

Synthetic repetitive extragenic palindromic (REP) sequence as an efficient mRNA stabilizer for protein production and metabolic engineering in prokaryotic cells

In prokaryotic cells, 3'-5' exonucleases can attenuate messenger RNA (mRNA) directionally from the direction of the 3'-5' untranslated region (UTR), and thus improving the stability of mRNAs without influencing normal cell growth and metabolism is a key challenge for protein production and metabolic engineering. Herein, we significantly improved mRNA stability by using synthetic repetitive extragenic palindromic (REP) sequences as an effective mRNA stabilizer in two typical prokaryotic microbes, namely, Escherichia coli for the production of cyclodextrin glucosyltransferase (CGTase) and Corynebacterium glutamicum for the production of N-acetylglucosamine (GlcNAc). First, we performed a high-throughput screen to select 4 out of 380 REP sequences generated by randomizing 6 nonconservative bases in the REP sequence designed as the degenerate base "N." Secondly, the REP sequence was inserted at several different positions after the stop codon of the CGTase-encoding gene. We found that mRNA stability was improved only when the space between the REP sequence and stop codon was longer than 12 base pairs (bp). Then, by reconstructing the spacer sequence and secondary structure of the REP sequence, a REP sequence with 8 bp in a stem-loop was obtained, and the CGTase activity increased from 210.6 to 291.5 U/ml. Furthermore, when this REP sequence was added to the 3'-UTR of glucosamine-6-phosphate N-acetyltransferase 1 ( GNA1), which is a gene encoding a key enzyme GNA1 in the GlcNAc synthesis pathway, the GNA1 activity was increased from 524.8 to 890.7 U/mg, and the GlcNAc titer was increased from 4.1 to 6.0 g/L in C. glutamicum. These findings suggest that the REP sequence plays an important function as an mRNA stabilizer in prokaryotic cells to stabilize its 3'-terminus of the mRNA by blocking the processing action of the 3'-5' exonuclease. Overall, this study provides new insight for the high-efficiency overexpression of target genes and pathway fine-tuning in bacteria.

Nanoparticle abraxane possesses impaired proliferation in A549 cells due to the underexpression of glucosamine 6-phosphate N-acetyltransferase 1 (GNPNAT1/GNA1).

Abraxane (Abr), a US Food and Drug Administration-approved albumin-bound nanoparticle applied for the treatment of non-small-cell lung cancer, has been reported to be more effective than paclitaxel (PTX). To further understand the molecular mechanisms that produce this superior drug efficacy of Abr, a quantitative proteomic approach has been applied to investigate the global protein expression profiles of lung cancer cell A549 treated with Abr and PTX. Only one protein, namely, glucosamine 6-phosphate N-acetyltransferase 1 (GNA1), showed significant differential expression (P<0.05) in the cutoff of 2.0 fold, suggesting that Abr can be used safely as a substitute for PTX. GNA1 is a key enzyme in the biosynthesis of uridine diphosphate-N-acetylglucosamine, which is an important donor substrate for N-linked glycosylation and has several important functions such as embryonic development and growth. Albumin plays a major role in the regulation of this protein. In summary, this study first shows that the superior drug effect of Abr is mainly due to the downregulation of GNA1, which causes proliferative delay and cell adhesion defect. It is also noteworthy that the deficiency of GNA1 might reduce insulin secretion which correlates with type 2 diabetes.

Stable Isotope Labeling with Amino Acids (SILAC)-Based Proteomics of Primary Human Kidney Cells Reveals a Novel Link between Male Sex Hormones and Impaired Energy Metabolism in Diabetic Kidney Disease

Male sex predisposes to many kidney diseases. Considering that androgens exert deleterious effects in a variety of cell types within the kidney, we hypothesized that dihydrotestosterone (DHT) would alter the biology of the renal tubular cell by inducing changes in the proteome. We employed stable isotope labeling with amino acids (SILAC) in an indirect spike-in fashion to accurately quantify the proteome in DHT- and 17β-estradiol (EST)-treated human proximal tubular epithelial cells (PTEC). Of the 5043 quantified proteins, 76 were differentially regulated. Biological processes related to energy metabolism were significantly enriched among DHT-regulated proteins. SILAC ratios of 3 candidates representing glycolysis, N-acetylglucosamine metabolism and fatty acid β-oxidation, namely glucose-6-phosphate isomerase (GPI), glucosamine-6-phosphate-N-acetyltransferase 1 (GNPNAT1), and mitochondrial trifunctional protein subunit alpha (HADHA), were verified in vitro. In vivo, renal GPI and HADHA protein expression was significantly increased in males. Furthermore, male sex was associated with significantly higher GPI, GNPNAT1, and HADHA kidney protein expression in two different murine models of diabetes. Enrichment analysis revealed a link between our DHT-regulated proteins and oxidative stress within the diabetic kidney. This finding was validated in vivo, as we observed increased oxidative stress levels in control and diabetic male kidneys, compared with females. This in depth quantitative proteomics study of human primary PTEC response to sex hormone administration suggests that male sex hormone stimulation results in perturbed energy metabolism in kidney cells, and that this perturbation results in increased oxidative stress in the renal cortex. The proteome-level changes associated with androgens may play a crucial role in the development of structural and functional changes in the diseased kidney. With our findings, we propose a possible link between diabetic and non-diabetic kidney disease progression and male sex hormone levels. Data are available via ProteomeXchange (https://www.ebi.ac.uk/pride/archive/) with identifier PXD003811.

Human acetyl-CoA:glucosamine-6-phosphate N-acetyltransferase 1 has a relaxed donor specificity and transfers acyl groups up to four carbons in length

Glucosamine-6-phosphate N-acetyltransferase1 (GNA1) catalyses the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to glucosamine-6-phosphate (GlcN6P) to form N-acetylglucosamine-6-phosphate (GlcNAc6P), which is an essential intermediate in UDP-GlcNAc biosynthesis. An analog of GlcNAc, N-butyrylglucosamine (GlcNBu) has shown healing properties for bone and articular cartilage in animal models of arthritis. The goal of this work was to examine whether GNA1 has the ability to transfer a butyryl group from butyryl-CoA to GlcN6P to form GlcNBu6P, which can then be converted to GlcNBu. We developed fluorescent and radioactive assays and examined the donor specificity of human GNA1. Acetyl, propionyl, n-butyryl, and isobutyryl groups were all transferred to GlcN6P, but isovaleryl-CoA and decanoyl-CoA did not serve as donor substrates. Site-specific mutants were produced to examine the role of amino acids potentially affecting the size and properties of the AcCoA binding pocket. All of the wild type and mutant enzymes showed activities of both acetyl and butyryl transfer and can therefore be used for the enzymatic synthesis of GlcNBu for biomedical applications.

Artificial Environments for the Co-Translational Stabilization of Cell-Free Expressed Proteins

An approach for designing individual expression environments that reduce or prevent protein aggregation and precipitation is described. Inefficient folding of difficult proteins in unfavorable translation environments can cause significant losses of overexpressed proteins as precipitates or inclusion bodies. A number of chemical chaperones including alcohols, polyols, polyions or polymers are known to have positive effects on protein stability. However, conventional expression approaches can use such stabilizing agents only post-translationally during protein extraction and purification. Proteins that already precipitate inside of the producer cells cannot be addressed. The open nature of cell-free protein expression systems offers the option to include single chemicals or cocktails of stabilizing compounds already into the expression environment. We report an approach for systematic screening of stabilizers in order to improve the solubility and quality of overexpressed proteins co-translationally. A comprehensive list of representative protein stabilizers from the major groups of naturally occurring chemical chaperones has been analyzed and their concentration ranges tolerated by cell-free expression systems have been determined. As a proof of concept, we have applied the method to improve the yield of proteins showing instability and partial precipitation during cell-free synthesis. Stabilizers that co-translationally improve the solubility and functional folding of human glucosamine 6-phosphate N-acetyltransferase have been identified and cumulative effects of stabilizers have been studied.

Cell-free expression of human glucosamine 6-phosphate N-acetyltransferase (HsGNA1) for inhibitor screening

Glucosamine 6-phosphate N-acetyltransferase (GNA1; EC 2.3.1.4) is required for the de novo synthesis of N-acetyl-d-glucosamine-6-phosphate (GlcNAc-6P), which is an essential precursor in Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) biosynthesis pathway. Therefore, GNA1 is indispensable for the viability of organisms. Here, a novel cell-free expression strategy was developed to efficiently produce large amounts of human GNA1(HsGNA1) and HsGNA1-sGFP for throughput inhibitor screening. The binding site of inhibitor glucose-6-phosphate (G6P) to hGNA was identified by simulated annealing. Subtle differences to the binding site of Aspergillius GNA1(AfGNA1) can be harnessed for inhibitor design. HsGNA1 may be also useful as an antimicrobial and chemotherapeutic target against cancer. Additionally HsGNA1 inhibitors/modulators can possibly be administered with other drugs in the next generation of personalized medicine.

A Missense Mutation in the Glucosamine-6-Phosphate N-Acetyltransferase–Encoding Gene Causes Temperature-Dependent Growth Defects and Ectopic Lignin Deposition in Arabidopsis

To study the regulatory mechanisms underlying lignin biosynthesis, we isolated and characterized lignescens (lig), a previously undescribed temperature-sensitive mutant of Arabidopsis thaliana that exhibits ectopic lignin deposition and growth defects under high-temperature conditions. The lig mutation was identified as a single base transition in GNA1 encoding glucosamine-6-phosphate N-acetyltransferase (GNA), a critical enzyme of UDP-N-acetylglucosamine (UDP-GlcNAc) biosynthesis. lig harbors a glycine-to-serine substitution at residue 68 (G68S) of GNA1. Enzyme activity assays of the mutant protein (GNA1(G68S)) showed its thermolability relative to the wild-type protein. The lig mutant exposed to the restrictive temperature contained a significantly smaller amount of UDP-GlcNAc than did the wild type. The growth defects and ectopic lignification of lig were suppressed by the addition of UDP-GlcNAc. Since UDP-GlcNAc is an initial sugar donor of N-glycan synthesis and impaired N-glycan synthesis is known to induce the unfolded protein response (UPR), we examined possible relationships between N-glycan synthesis, UPR, and the lig phenotype. N-glycans were reduced and LUMINAL BINDING PROTEIN3, a typical UPR gene, was expressed in lig at the restrictive temperature. Furthermore, treatment with UPR-inducing reagents phenocopied the lig mutant. Our data collectively suggest that impairment of N-glycan synthesis due to a shortage of UDP-GlcNAc leads to ectopic lignin accumulation, mostly through the UPR.

Structural and biochemical characterization of a trapped coenzyme A adduct ofCaenorhabditis elegansglucosamine-6-phosphateN-acetyltransferase 1

Glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) produces GlcNAc-6-phosphate from GlcN-6-phosphate and acetyl coenzyme A. Early mercury-labelling experiments implicated a conserved cysteine in the reaction mechanism, whereas recent structural data appear to support a mechanism in which this cysteine plays no role. Here, two crystal structures of Caenorhabditis elegans GNA1 are reported, revealing an unusual covalent complex between this cysteine and the coenzyme A product. Mass-spectrometric and reduction studies showed that this inactive covalent complex can be reactivated through reduction, yet mutagenesis of the cysteine supports a previously reported bi-bi mechanism. The data unify the apparently contradictory earlier reports on the role of a cysteine in the GNA1 active site.

Crystal structure and functional characterization of a glucosamine-6-phosphate N-acetyltransferase from Arabidopsis thaliana

GlcNAc (N-acetylglucosamine) is an essential part of the glycan chain in N-linked glycoproteins. It is a building block for polysaccharides such as chitin, and several glucosaminoglycans and proteins can be O-GlcNAcylated. The deacetylated form, glucosamine, is an integral part of GPI (glycosylphosphatidylinositol) anchors. Both are incorporated into polymers by glycosyltransferases that utilize UDP-GlcNAc. This UDP-sugar is synthesized in a short pathway comprising four steps starting from fructose 6-phosphate. GNA (glucosamine-6-phosphate N-acetyltransferase) catalyses the second of these four reactions in the de novo synthesis in eukaryotes. A phylogenetic analysis revealed that only one GNA isoform can be found in most of the species investigated and that the most likely Arabidopsis candidate is encoded by the gene At5g15770 (AtGNA). qPCR (quantitative PCR) revealed the ubiquitous expression of AtGNA in all organs of Arabidopsis plants. Heterologous expression of AtGNA showed that it is highly active between pH 7 and 8 and at temperatures of 30-40°C. It showed Km values of 231 μM for glucosamine 6-phosphate and 33 μM for acetyl-CoA respectively and a catalytic efficiency comparable with that of other GNAs characterized. The solved crystal structure of AtGNA at a resolution of 1.5 Å (1 Å=0.1 nm) revealed a very high structural similarity to crystallized GNA proteins from Homo sapiens and Saccharomyces cerevisiae despite less well conserved protein sequence identity.

Characterization, localization, essentiality, and high-resolution crystal structure of glucosamine 6-phosphate N-acetyltransferase from Trypanosoma brucei.

A gene predicted to encode Trypanosoma brucei glucosamine 6-phosphate N-acetyltransferase (TbGNA1; EC 2.3.1.4) was cloned and expressed in Escherichia coli. The recombinant protein was enzymatically active, and its high-resolution crystal structure was obtained at 1.86 Å. Endogenous TbGNA1 protein was localized to the peroxisome-like microbody, the glycosome. A bloodstream-form T. brucei GNA1 conditional null mutant was constructed and shown to be unable to sustain growth in vitro under nonpermissive conditions, demonstrating that there are no metabolic or nutritional routes to UDP-GlcNAc other than via GlcNAc-6-phosphate. Analysis of the protein glycosylation phenotype of the TbGNA1 mutant under nonpermissive conditions revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite and that the glycosylation profile of the principal parasite surface coat component, the variant surface glycoprotein (VSG), was modified. The significance of results and the potential of TbGNA1 as a novel drug target for African sleeping sickness are discussed.

Acceptor substrate binding revealed by crystal structure of human glucosamine-6-phosphate N-acetyltransferase 1

Glucosamine-6-phosphate (GlcN6P) N-acetyltransferase 1 (GNA1) is a key enzyme in the pathway toward biosynthesis of UDP- N-acetylglucosamine, an important donor substrate for N-linked glycosylation. GNA1 catalyzes the formation of N-acetylglucosamine-6-phosphate (GlcNAc6P) from acetyl-CoA (AcCoA) and the acceptor substrate GlcN6P. Here, we report crystal structures of human GNA1, including apo GNA1, the GNA1-GlcN6P complex and an E156A mutant. Our work showed that GlcN6P binds to GNA1 without the help of AcCoA binding. Structural analyses and mutagenesis studies have shed lights on the charge distribution in the GlcN6P binding pocket, and an important role for Glu156 in the substrate binding. Hence, these findings have broadened our knowledge of structural features required for the substrate affinity of GNA1. Structured summary MINT: 6700314: GNA1 (uniprotkb: Q96EK6) and GNA1 (uniprotkb: Q96EK6) bind (MI: 0407) by X-ray crystallography (MI: 0114)

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 230 nm 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.

Mosquito glucosamine-6-phosphate N-acetyltransferase: cDNA, gene structure and enzyme kinetics

Mosquito midgut epithelial cells secrete digestive enzymes as well as components of the peritrophic matrix in response to blood-feeding. The peritrophic matrix is composed of proteins, glycoproteins and chitin fibrils in a proteoglycan matrix and may function to protect the midgut epithelium from mechanical damage and insult from pathogens and toxins. Chitin biosynthesis takes place via the hexosamine pathway converting fructose-6-phsophate to UDP- N-acetylglucosamine, which is then polymerized to chitin by chitin synthase. Glucosamine-6-phosphate N-acetyltransferase (GNA) is one of the hexosamine pathway enzymes and catalyzes the transfer of the acetyl group from acetyl-CoA to the primary amine of glucosamine-6-phosphate. We cloned and sequenced the GNA cDNA, gene ( AeGna) and its putative promoter regions from Aedes aegypti. AeGna consists of five exons and four introns and lacks a TATA box near the transcription start site. The AeGna cDNA is 1.3 kb in length and the predicted protein is approximately 23.6 kDa. The amino acid sequence of AeGna has high homology to its orthologues. AeGna mRNA is constitutively expressed in all developmental stages and blood-feeding causes no obvious effect on levels of AeGna transcript in the midgut. The Km value of recombinant GNA for glucosamine-6-phosphate was 330 μM and the Km for acetyl-CoA was 500 μM.

Transcription regulation is demonstrated for five key enzymes in Giardia intestinalis cyst wall polysaccharide biosynthesis.

The cyst wall of Giardia intestinalis contains proteins and a novel N-acetylgalactosamine (GalNAc) polysaccharide, which is its major constituent. GalNAc is not present in growing trophozoites, but is synthesized during encystment via an inducible pathway of enzymes that produce UDP-GalNAc from fructose 6-phosphate. This report focuses on the regulation of these enzymes and thus the genes for glucosamine 6-phosphate N-acetyltransferase (GNA), phosphoacetylglucosamine mutase (AGM), UDP-N-acetylglucosamine pyrophosphorylase (UAP), and UDP-N-acetylglucosamine 4-epimerase (UAE) were cloned and expressed in Escherichia coli. Each of these expressed enzymes had the predicted activity and was used to generate antibodies. Northern and Western blot analyses demonstrated that both the mRNA and protein levels for all of these enzymes increase during encystment. Nuclear run-on assays of these and the previously analyzed glucosamine 6-phosphate deaminase (GNP; glucosamine 6-P isomerase) showed that all of the genes responsible for UDP-GalNAc synthesis during encystment are induced at the transcription level.