A novel variant in GNPNAT1 gene causing a spondylo-epi-metaphyseal dysplasia resembling PGM3-Desbuquois like dysplasia.

Spondylo-epi-metaphyseal dysplasias (SEMDs) are a clinically and genetically heterogeneous group of skeletal dysplasias characterized by short stature and abnormal modeling of the spine and long bones. A novel form of rhizomelic skeletal dysplasia, Ain-Naz type, associated with a homozygous variant in GNPNAT1 was recently identified. Herein, we report an Egyptian patient, offspring of consanguineous parents, who presented with a severe form of unclassified SEMD. Whole exome sequencing identified a novel homozygous variant in exon 3, c.77T>G, (p.Phe26Cys) in GNPNAT1, that was confirmed by Sanger sequencing and both parents were found to be heterozygous for the identified variant. Main features included severe short stature, rhizomelic limb shortening, and wide flared metaphysis. Short broad long bones, brachydactyly, delayed epiphyseal ossification of long bones, advanced bone age, and immunodeficiency were additional findings expanding the clinical phenotype described in the previously reported family. We conclude that variants in the GNPNAT1 gene cause an autosomal recessive form of SEMD resembling Desbuquois like dysplasia caused by PGM3, which is involved in the same pathway as GNPNAT1.

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

BACKGROUND: Studies exploring molecular mechanisms underlying congenital skeletal disorders have revealed novel regulators of skeletal homeostasis and shown protein glycosylation to play an important role. OBJECTIVE: To identify the genetic cause of rhizomelic skeletal dysplasia in a consanguineous Pakistani family. METHODS: Clinical investigations were carried out for four affected individuals in the recruited family. Whole genome sequencing (WGS) was completed using DNA from two affected and two unaffected individuals from the family. Sequencing data were processed, filtered and analysed. In silico analyses were performed to predict the effects of the candidate variant on the protein structure and function. Small interfering RNAs (siRNAs) were used to study the effect of Gnpnat1 gene knockdown in primary rat chondrocytes. RESULTS: The patients presented with short stature due to extreme shortening of the proximal segments of the limbs. Radiographs of one individual showed hip dysplasia and severe platyspondyly. WGS data analyses identified a homozygous missense variant c.226G>A; p.(Glu76Lys) in GNPNAT1, segregating with the disease. Glucosamine 6-phosphate N-acetyltransferase, encoded by the highly conserved gene GNPNAT1, is one of the enzymes required for synthesis of uridine diphosphate N-acetylglucosamine, which participates in protein glycosylation. Knockdown of Gnpnat1 by siRNAs decreased cellular proliferation and expression of chondrocyte differentiation markers collagen type 2 and alkaline phosphatase, indicating that Gnpnat1 is important for growth plate chondrocyte proliferation and differentiation. CONCLUSIONS: This study describes a novel severe skeletal dysplasia associated with a biallelic, variant in GNPNAT1. Our data suggest that GNPNAT1 is important for growth plate chondrogenesis.

Diverse Mechanisms Lead to Common Dysfunction of Striatal Cholinergic Interneurons in Distinct Genetic Mouse Models of Dystonia.

Clinical and experimental data indicate striatal cholinergic dysfunction in dystonia, a movement disorder typically resulting in twisted postures via abnormal muscle contraction. Three forms of isolated human dystonia result from mutations in the TOR1A (DYT1), THAP1 (DYT6), and GNAL (DYT25) genes. Experimental models carrying these mutations facilitate identification of possible shared cellular mechanisms. Recently, we reported elevated extracellular striatal acetylcholine by in vivo microdialysis and paradoxical excitation of cholinergic interneurons (ChIs) by dopamine D2 receptor (D2R) agonism using ex vivo slice electrophysiology in Dyt1ΔGAG/+ mice. The paradoxical excitation was caused by overactive muscarinic receptors (mAChRs), leading to a switch in D2R coupling from canonical Gi/o to noncanonical ß-arrestin signaling. We sought to determine whether these mechanisms in Dyt1ΔGAG/+ mice are shared with Thap1C54Y/+ knock-in and Gnal+/- knock-out dystonia models and to determine the impact of sex. We found Thap1C54Y/+ mice of both sexes have elevated extracellular striatal acetylcholine and D2R-induced paradoxical ChI excitation, which was reversed by mAChR inhibition. Elevated extracellular acetylcholine was absent in male and female Gnal+/- mice, but the paradoxical D2R-mediated ChI excitation was retained and only reversed by inhibition of adenosine A2ARs. The Gi/o-preferring D2R agonist failed to increase ChI excitability, suggesting a possible switch in coupling of D2Rs to ß-arrestin, as seen previously in a DYT1 model. These data show that, whereas elevated extracellular acetylcholine levels are not always detected across these genetic models of human dystonia, the D2R-mediated paradoxical excitation of ChIs is shared and is caused by altered function of distinct G-protein-coupled receptors.SIGNIFICANCE STATEMENT Dystonia is a common and often disabling movement disorder. The usual medical treatment of dystonia is pharmacotherapy with nonselective antagonists of muscarinic acetylcholine receptors, which have many undesirable side effects. Development of new therapeutics is a top priority for dystonia research. The current findings, considered in context with our previous investigations, establish a role for cholinergic dysfunction across three mouse models of human genetic dystonia: DYT1, DYT6, and DYT25. The commonality of cholinergic dysfunction in these models arising from diverse molecular etiologies points the way to new approaches for cholinergic modulation that may be broadly applicable in dystonia.

Combinatorial pathway enzyme engineering and host engineering overcomes pyruvate overflow and enhances overproduction of N-acetylglucosamine in Bacillus subtilis.

BACKGROUND: Glucosamine-6-phosphate N-acetyltransferase (GNA1) is the key enzyme that causes overproduction of N-acetylglucosamine in Bacillus subtilis. Previously, we increased GlcNAc production by promoting the expression of GNA1 from Caenorhabditis elegans (CeGNA1) in an engineered B. subtilis strain BSGN12. In this strain overflow metabolism to by-products acetoin and acetate had been blocked by mutations, however pyruvate accumulated as an overflow metabolite. Although overexpression of CeGNA1 drove carbon flux from pyruvate to the GlcNAc synthesis pathway and decreased pyruvate accumulation, the residual pyruvate reduced the intracellular pH, resulting in inhibited CeGNA1 activity and limited GlcNAc production. RESULTS: In this study, we attempted to further overcome pyruvate overflow by enzyme engineering and host engineering for enhanced GlcNAc production. To this end, the key enzyme CeGNA1 was evolved through error-prone PCR under pyruvate stress to enhance its catalytic activity. Then, the urease from Bacillus paralicheniformis was expressed intracellularly to neutralize the intracellular pH, making it more robust in growth and more efficient in GlcNAc production. It was found that the activity of mutant CeGNA1 increased by 11.5% at pH 6.5-7.5, with the catalytic efficiency increasing by 27.5% to 1.25 s-1 µM-1. Modulated expression of urease increased the intracellular pH from 6.0 to 6.8. The final engineered strain BSGN13 overcame pyruvate overflow, produced 25.6 g/L GlcNAc with a yield of 0.43 g GlcNAc/g glucose in a shake flask fermentation and produced 82.5 g/L GlcNAc with a yield of 0.39 g GlcNAc/g glucose by fed-batch fermentation, which was 1.7- and 1.2-times, respectively, of the yield achieved previously. CONCLUSIONS: This study highlights a strategy that combines pathway enzyme engineering and host engineering to resolve overflow metabolism in B. subtilis for the overproduction of GlcNAc. By means of modulated expression of urease reduced pyruvate burden, conferred bacterial survival fitness, and enhanced GlcNAc production, all of which improved our understanding of co-regulation of cell growth and metabolism to construct more efficient B. subtilis cell factories.

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.

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.

Characterization of the amino acid residues mediating the unique amino-sugar-1-phosphate acetyltransferase activity of the archaeal ST0452 protein.

The ST0452 protein from the thermophilic archaean Sulfolobus tokodaii has been identified as an enzyme with multiple sugar-1-phosphate nucleotidylyltransferase and amino-sugar-1-phosphate acetyltransferase (amino-sugar-1-P AcTase) activities. Analysis of the protein showed that in addition to glucosamine-1-phosphate (GlcN-1-P) AcTase activity, it possesses unique galactosamine-1-phosphate (GalN-1-P) AcTase activity not detected in any other proteins. Comparison of the crystal structures of the ST0452 protein and GlmU from Escherichia coli (EcGlmU), which possesses only GlcN-1-P AcTase activity, showed that the overall sequence identity between these two proteins is less than 25 %, but the amino acid residues predicted to comprise the catalytic center of EcGlmU are conserved in the ST0452 protein. To understand the molecular mechanism by which the ST0452 amino-sugar-1-P AcTase activity recognizes two independent substrates, several ST0452 substitution and truncation mutant proteins were constructed and analyzed. We found that His308 is essential for both GalN-1-P and GlcN-1-P AcTase activities, whereas Tyr311 and Asn331 are important only for the GalN-1-P AcTase activity. In addition, deletion of the C-terminal 5 or 11 residues showed that the 11-residue C-terminal region exerts a modest stimulatory effect on GalN-1-P AcTase activity but dramatically suppresses GlcN-1-P AcTase activity. This region also appears to make an important contribution to the thermostability of the entire ST0452 protein. Systematic deletions from the C-terminus also demonstrated that the C-terminal region with the ß-helix structure has an important role mediating the trimerization of the ST0452 protein. This is the first report of an analysis of a thermostable archaeal enzyme exhibiting multiple amino-sugar-1-P AcTase activities.

The importance of ether-phospholipids: a view from the perspective of mouse models.

Ether-phospholipids represent an important group of phospholipids characterized by an alkyl or an alkenyl bond at the sn-1 position of the glycerol backbone. Plasmalogens are the most abundant form of alkenyl-glycerophospholipids, and their synthesis requires functional peroxisomes. Defects in the biosynthesis of plasmalogens are the biochemical hallmark of the human peroxisomal disorder Rhizomelic Chondrodysplasia Punctata (RCDP), which is characterized by defects in eye, bone and nervous tissue. The generation and characterization of mouse models with defects in plasmalogen levels have significantly advanced our understanding of the role and importance of plasmalogens as well as pathogenetic mechanisms underlying RCDP. A review of the current mouse models and the description of the combined knowledge gathered from the histopathological and biochemical studies is presented and discussed. Further characterization of the role and functions of plasmalogens will contribute to the elucidation of disease pathogenesis in peroxisomal and non-peroxisomal disorders. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.

Pathway engineering of Bacillus subtilis for microbial production of N-acetylglucosamine.

Glucosamine (GlcN) and its acetylated derivative, N-acetylglucosamine (GlcNAc), are widely used in nutraceutical and pharmaceutical industries. Currently, GlcN and GlcNAc are mainly produced by hydrolysis from crab and shrimp shells, which can cause severe environmental pollution and carries the potential risk of allergic reactions. In this study, we attempted to achieve microbial production of GlcNAc by pathway engineering of Bacillus subtilis 168. Specifically, glmS (encoding GlcN-6-phosphate synthase) from B. subtilis 168 and GNA1 (encoding GlcNAc-6-phosphate N-acetyltransferase) from Saccharomyces cerevisiae S288C were firstly co-overexpressed in B. subtilis; the level of GlcNAc reached 240mg/L in shake flask culture. Next, nagP, encoding the GlcNAc-specific enzyme of phosphotransferase system, was deleted to block the importation of extracellular GlcNAC, thus improving GlcNAc production to 615mg/L in shake flask culture. Then, nagA (encoding GlcNAc-6-phosphate deacetylase), gamA (encoding GlcN-6-phosphate deaminase), and nagB (encoding GlcN-6-phosphate deaminase) were deleted to block the catabolism of intracellular GlcNAc, thereby further increasing the GlcNAc titer to 1.85g/L in shake flask culture. Finally, microbial production of GlcNAc by the engineered B. subtilis 168 was conducted in a 3-L fed-batch bioreactor, and the GlcNAc titer reached 5.19g/L, which was 2.8-fold of that in shake flask culture. This is the first report regarding the pathway engineering of B. subtilis for microbial production of GlcNAc, and provides a good starting point for further metabolic engineering to achieve the industrial production of GlcNAc by a generally regarded as safe strain.

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.

Engineering of an N-acetylneuraminic acid synthetic pathway in Escherichia coli.

N-acetylneuraminic acid (NeuAc) has recently drawn much attention owing to its wide applications in many aspects. Besides extraction from natural materials, production of NeuAc was recently focused on enzymatic synthesis and whole-cell biocatalysis. In this study, we designed an artificial NeuAc biosynthetic pathway through intermediate N-acetylglucosamine 6-phosphate in Escherichia coli. In this pathway, N-acetylglucosamine 2-epimerase (slr1975) and glucosamine-6-phosphate acetyltransferase (GNA1) were heterologously introduced into E. coli from Synechocystis sp. PCC6803 and Saccharomyces cerevisiae EBY100, respectively. By derepressing the feedback inhibition of glucosamine-6-phosphate synthase, increasing the accumulation of N-acetylglucosamine and pyruvate, and blocking the catabolism of NeuAc, we were able to produce 1.62 g l⁻¹ NeuAc in recombinant E. coli directly from glucose. The NeuAc yield reached 7.85g l⁻¹ in fed-batch fermentation. This process offered an efficient fermentative method to produce NeuAc in microorganisms using glucose as carbon source and can be optimized for further improvement.

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.

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.

Glucosamine-6-phosphate N-acetyltransferase gene silencing by parental RNA interference in rice leaf folder, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae).

Parental RNAi (pRNAi) is a response of RNA interference in which treated insect pests progenies showed a gene silencing phenotypes. pRNAi of CmGNA gene has been studied in Cnaphalocrocis medinalis via injection. Our results showed significant reduction in ovulation per female that was 26% and 35.26% in G1 and G2 generations, respectively. Significant reduction of hatched eggs per female were observed 23.53% and 45.26% as compared to control in G1-G2 generations, respectively. We also observed the significant variation in the sex ratio between female (40% and 53%) in G1-G2 generations, and in male (65%) in G1 generation as compared to control. Our results also demonstrated the significant larval mortality (63% and 55%) and pupal mortality (55% and 41%), and significant reduction of mRNA expression level in G1 and G2 generations. Our findings have confirmed that effectiveness of pRNAi induced silencing on the CmGNA target gene in G1-G2 generations of C. medinalis. These results suggested the potential role of pRNAi in insect pest resistance management strategies.

A second individual with rhizomelic spondyloepimetaphyseal dysplasia and homozygous variant in GNPNAT1.

Spondyloepimetaphyseal dysplasias (SEMDs) belong to a clinically and genetically heterogeneous group of inherited skeletal disorders defined by a defect in the growth and shape of vertebrae, epiphyses and metaphyses. Rhizomelic SEMD is characterized by a disproportionate small stature caused by severe shortening and deformation of the limbs' proximal bones, with the cranio-facial sphere unaffected. We report a second individual, an 8-year-old girl, with autosomal recessive rhizomelic SEMD associated with a homozygous exonic missense variant, c.226G > A p.(Glu76Lys), in GNPNAT1 identified by trio genome sequencing. Our data corroborate the recent findings of Ain et al. and further delineate the clinical and radiographic features of this form of SEMD associated with rhizomelic dysplasia while outlining a potential hotspot in this newly described genetic disorder.

Potential role of glucosamine-phosphate N-acetyltransferase 1 in the development of lung adenocarcinoma.

Glucosamine-phosphate N-acetyltransferase 1 (GNPNAT1) is a key enzyme associated with glucose metabolism and uridine diphosphate-N-acetylglucosamine biosynthesis. Abnormal GNPNAT1 expression might be associated with carcinogenesis. We analyzed multiple lung adenocarcinoma (LUAD) gene expression databases and verified GNPNAT1 higher expression in LUAD tumor tissues than in normal tissues. Moreover, we analyzed the survival relationship between LUAD patients' clinical status and GNPNAT1 expression, and found higher GNPNAT1 expression in LUAD patients with unfavorable prognosis. We built GNPNAT1 gene co-expression networks and further annotated the co-expressed genes' Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and various associated regulatory factors. These co-expression genes' functional networks mainly participate in chromosome segregation, RNA metabolic process, and RNA transport. We analyzed GNPNAT1 genetic alterations and co-occurrence networks, and the functional networks of these genes showed that GNPNAT1 participates in multiple steps of cell cycle transition and in the development of some cancers. We assessed the correlation between GNPNAT1 expression and cancer immune infiltrates and showed that GNPNAT1 expression is correlated with several immune cells, chemokines, and immunomodulators in LUAD. We found that GNPNAT1 correlates with LUAD development and prognosis, laying a foundation for further research, especially in immunotherapy.

Plasmodium falciparum Apicomplexan-Specific Glucosamine-6-Phosphate N-Acetyltransferase Is Key for Amino Sugar Metabolism and Asexual Blood Stage Development.

UDP-N-acetylglucosamine (UDP-GlcNAc), the main product of the hexosamine biosynthetic pathway, is an important metabolite in protozoan parasites since its sugar moiety is incorporated into glycosylphosphatidylinositol (GPI) glycolipids and N- and O-linked glycans. Apicomplexan parasites have a hexosamine pathway comparable to other eukaryotic organisms, with the exception of the glucosamine-phosphate N-acetyltransferase (GNA1) enzymatic step that has an independent evolutionary origin and significant differences from nonapicomplexan GNA1s. By using conditional genetic engineering, we demonstrate the requirement of GNA1 for the generation of a pool of UDP-GlcNAc and for the development of intraerythrocytic asexual Plasmodium falciparum parasites. Furthermore, we present the 1.95 Å resolution structure of the GNA1 ortholog from Cryptosporidium parvum, an apicomplexan parasite which is a leading cause of diarrhea in developing countries, as a surrogate for P. falciparum GNA1. The in-depth analysis of the crystal shows the presence of specific residues relevant for GNA1 enzymatic activity that are further investigated by the creation of site-specific mutants. The experiments reveal distinct features in apicomplexan GNA1 enzymes that could be exploitable for the generation of selective inhibitors against these parasites, by targeting the hexosamine pathway. This work underscores the potential of apicomplexan GNA1 as a drug target against malaria.IMPORTANCE Apicomplexan parasites cause a major burden on global health and economy. The absence of treatments, the emergence of resistances against available therapies, and the parasite's ability to manipulate host cells and evade immune systems highlight the urgent need to characterize new drug targets to treat infections caused by these parasites. We demonstrate that glucosamine-6-phosphate N-acetyltransferase (GNA1), required for the biosynthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), is essential for P. falciparum asexual blood stage development and that the disruption of the gene encoding this enzyme quickly causes the death of the parasite within a life cycle. The high-resolution crystal structure of the GNA1 ortholog from the apicomplexan parasite C. parvum, used here as a surrogate, highlights significant differences from human GNA1. These divergences can be exploited for the design of specific inhibitors against the malaria parasite.

Identification Six Metabolic Genes as Potential Biomarkers for Lung Adenocarcinoma.

Metabolic genes have been reported to act as crucial roles in tumor progression. Lung adenocarcinoma (LUAD) is one of the most common cancers worldwide. This study aimed to predict the potential mechanism and novel markers of metabolic signature in LUAD. The gene expression profiles and the clinical parameters were obtained from The Cancer Genome Atlas-Lung adenocarcinoma (TCGA-LUAD) and Gene Expression Omnibus data set (GSE72094). A total of 105 differentially expressed metabolic genes of intersect expression in TCGA-LUAD and GSE72094 were screened by R language. Univariate Cox regression model found 18 survival-related genes and then the least absolute shrinkage and selection operator model was successfully constructed. Six significant genes prognostic model was validated though independent prognosis analysis. The model revealed high values for prognostic biomarkers by time-dependent receiver operating characteristic (ROC) analysis, risk score, Heatmap, and nomogram. In addition, Gene Set Enrichment Analysis showed that multiplex metabolism pathways correlated with LUAD. Furthermore, we found the six genes aberrantly expressed in LUAD samples. Our study showed that metabolism pathways play important roles in LUAD progression. The six metabolic genes could predict potential prognostic and diagnostic biomarkers in LUAD.

Structural and kinetic differences between human and Aspergillus fumigatus D-glucosamine-6-phosphate N-acetyltransferase.

Aspergillus fumigatus is the causative agent of aspergillosis, a frequently invasive colonization of the lungs of immunocompromised patients. GNA1 (D-glucosamine-6-phosphate N-acetyltransferase) catalyses the acetylation of GlcN-6P (glucosamine-6-phosphate) to GlcNAc-6P (N-acetylglucosamine-6-phosphate), a key intermediate in the UDP-GlcNAc biosynthetic pathway. Gene disruption of gna1 in yeast and Candida albicans has provided genetic validation of the enzyme as a potential target. An understanding of potential active site differences between the human and A. fumigatus enzymes is required to enable further work aimed at identifying selective inhibitors for the fungal enzyme. In the present study, we describe crystal structures of both human and A. fumigatus GNA1, as well as their kinetic characterization. The structures show significant differences in the sugar-binding site with, in particular, several non-conservative substitutions near the phosphate-binding pocket. Mutagenesis targeting these differences revealed drastic effects on steady-state kinetics, suggesting that the differences could be exploitable with small-molecule inhibitors.

Combinatorial Fine-Tuning of GNA1 and GlmS Expression by 5'-Terminus Fusion Engineering Leads to Overproduction of N-Acetylglucosamine in Bacillus subtilis.

Glucosamine-6-phosphate N-acetyltransferase (GNA1) that catalyzes acetyl transfer from acetyl-coenzyme A to glucosamine-6-phosphate (GlcN-6P), and glutamine-fructose-6-phosphate aminotransferase (GlmS) that catalyzes the formation of GlcN-6P from fructose-6-phosphate (Fru-6P), are two key enzymes in Bacillus subtilis for the bioproduction of N-acetylglucosamine (GlcNAc), a nutraceutical that has various applications in healthcare. In this study, the expression of GNA1 and GlmS is fine-tuned by 5'-terminus fusion engineering to improve GlcNAc production. Specifically, the expression level of GNA1 is enhanced at the translational level via fusion of an epitope tag to the 5'-terminus of GNA1 gene and ribosome binding site (RBS) sequence engineering. Next, enhanced expression of GlmS is achieved at the transcriptional and translational levels by fusing an mRNA stabilizer to the 5'-terminus of GlmS gene. Under the control of GNA1 (fusion with cMyc tag and with the optimum RBS M-Rm) and GlmS (fusion with mRNA stabilizer ΔermC+14/7A), the GlcNAc titer and yield in the shake flask increase to 18.5 g L-1 and 0.37 g GlcNAc/g glucose, which are 2.9-fold and 2.3-fold that of the control, respectively. This synthetic pathway fine-tuning method at the transcriptional and translational levels by combinatorial modulation of regulatory elements, including epitope tag, RBS sequence, and mRNA stabilizer, might represent a general and effective approach for the construction of microbial cell factories.