Optimization of Metabolic Oligosaccharide Engineering with Ac4GalNAlk and Ac4GlcNAlk by an Engineered Pyrophosphorylase.

Metabolic oligosaccharide engineering (MOE) has fundamentally contributed to our understanding of protein glycosylation. Efficient MOE reagents are activated into nucleotide-sugars by cellular biosynthetic machineries, introduced into glycoproteins and traceable by bioorthogonal chemistry. Despite their widespread use, the metabolic fate of many MOE reagents is only beginning to be mapped. While metabolic interconnectivity can affect probe specificity, poor uptake by biosynthetic salvage pathways may impact probe sensitivity and trigger side reactions. Here, we use metabolic engineering to turn the weak alkyne-tagged MOE reagents Ac4GalNAlk and Ac4GlcNAlk into efficient chemical tools to probe protein glycosylation. We find that bypassing a metabolic bottleneck with an engineered version of the pyrophosphorylase AGX1 boosts nucleotide-sugar biosynthesis and increases bioorthogonal cell surface labeling by up to two orders of magnitude. A comparison with known azide-tagged MOE reagents reveals major differences in glycoprotein labeling, substantially expanding the toolbox of chemical glycobiology.

NDP-rhamnose biosynthesis and rhamnosyltransferases: building diverse glycoconjugates in nature.

Rhamnose is an important 6-deoxy sugar present in many natural products, glycoproteins, and structural polysaccharides. Whilst predominantly found as the l-enantiomer, instances of d-rhamnose are also found in nature, particularly in the Pseudomonads bacteria. Interestingly, rhamnose is notably absent from humans and other animals, which poses unique opportunities for drug discovery targeted towards rhamnose utilizing enzymes from pathogenic bacteria. Whilst the biosynthesis of nucleotide-activated rhamnose (NDP-rhamnose) is well studied, the study of rhamnosyltransferases that synthesize rhamnose-containing glycoconjugates is the current focus amongst the scientific community. In this review, we describe where rhamnose has been found in nature, as well as what is known about TDP-ß-l-rhamnose, UDP-ß-l-rhamnose, and GDP-α-d-rhamnose biosynthesis. We then focus on examples of rhamnosyltransferases that have been characterized using both in vivo and in vitro approaches from plants and bacteria, highlighting enzymes where 3D structures have been obtained. The ongoing study of rhamnose and rhamnosyltransferases, in particular in pathogenic organisms, is important to inform future drug discovery projects and vaccine development.

Repetitive Batch Mode Facilitates Enzymatic Synthesis of the Nucleotide Sugars UDP-Gal, UDP-GlcNAc, and UDP-GalNAc on a Multi-Gram Scale.

The availability of nucleotide sugars is considered as bottleneck for Leloir-glycosyltransferases mediated glycan synthesis. A breakthrough for the synthesis of nucleotide sugars is the development of salvage pathway like enzyme cascades with high product yields from affordable monosaccharide substrates. In this regard, the authors aim at high enzyme productivities of these cascades by a repetitive batch approach. The authors report here for the first time that the exceptional high enzyme cascade stability facilitates the synthesis of Uridine-5'-diphospho-α-d-galactose (UDP-Gal), Uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), and Uridine-5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) in a multi-gram scale by repetitive batch mode. The authors obtained 12.8 g UDP-Gal through a high mass based total turnover number (TTNmass ) of 494 [gproduct /genzyme ] and space-time-yield (STY) of 10.7 [g/L*h]. Synthesis of UDP-GlcNAc in repetitive batch mode gave 11.9 g product with a TTNmass of 522 [gproduct /genzyme ] and a STY of 9.9 [g/L*h]. Furthermore, the scale-up to a 200 mL scale using a pressure operated concentrator was demonstrated for a UDP-GalNAc producing enzyme cascade resulting in an exceptional high STY of 19.4 [g/L*h] and 23.3 g product. In conclusion, the authors demonstrate that repetitive batch mode is a versatile strategy for the multi-gram scale synthesis of nucleotide sugars by stable enzyme cascades.

Stepwise Synthesis of Quercetin Bisglycosides Using Engineered Escherichia coli.

Synthesis of flavonoid glycoside is difficult due to diverse hydroxy groups in flavonoids and sugars. As such, enzymatic synthesis or biotransformation is an approach to solve this problem. In this report, we used stepwise biotransformation to synthesize two quercetin bisglycosides (quercetin 3-O-glucuronic acid 7-O-rhamnoside [Q-GR] and quercetin 3-O-arabinose-7-O-rhamnoside [Q-AR]) because quercetin O-rhamnosides contain antiviral activity. Two sequential enzymatic reactions were required to synthesize these flavonoid glycosides. We first synthesized quercetin 3-O-glucuronic acid [Q-G], and quercetin 3-O-arabinose-[Q-A] from quercetin using E. coli harboring specific uridine diphopsphate glycosyltransferase (UGT) and genes for UDP-glucuronic acid and UDP-arabinose, respectively. With each quercetin 3-O-glycoside, rhamnosylation using E. coli harboring UGT and the gene for UDP-rhamnose was conducted. This approach resulted in the production of 44.8 mg/l Q-GR and 45.1 mg/l Q-AR. This stepwise synthesis could be applicable to synthesize various natural product derivatives in case that the final yield of product was low due to the multistep reaction in one cell or when sequential synthesis is necessary in order to reduce the synthesis of byproducts.

Involvement of Three CsRHM Genes from Camellia sinensis in UDP-Rhamnose Biosynthesis.

UDP-Rhamnose synthase (RHM), the branch-point enzyme controlling the nucleotide sugar interconversion pathway, converts UDP-d-glucose into UDP-rhamnose. As a rhamnose residue donor, UDP-l-rhamnose is essential for the biosynthesis of pectic polysaccharides and secondary metabolites in plants. In this study, three CsRHM genes from tea plants ( Camellia sinensis) were cloned and characterized. Enzyme assays showed that three recombinant proteins displayed RHM activity and were involved in the biosynthesis of UDP-rhamnose in vitro. The transcript profiles, metabolite profiles, and mucilage location suggest that the three CsRHM genes likely contribute to UDP-rhamnose biosynthesis and may be involved in primary wall formation in C. sinensis. These analyses of CsRHM genes and metabolite profiles provide a comprehensive understanding of secondary metabolite biosynthesis and regulation in tea plants. Moreover, our results can be applied for the synthesis of the secondary metabolite rhamnoside in future studies.

The Metabolic Chemical Reporter 6-Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity of O-GlcNAc Transferase and Catalyzes the Discovery of Intracellular Protein Modification by O-Glucose.

Metabolic chemical reporters of glycosylation in combination with bioorthogonal reactions have been known for two decades and have been used by many different research laboratories for the identification and visualization of glycoconjugates. More recently, however, they have begun to see utility for the investigation of cellular metabolism and the tolerance of biosynthetic enzymes and glycosyltransferases to different sugars. Here, we take this concept one step further by using the metabolic chemical reporter 6-azido-6-deoxy-glucose (6AzGlc). We show that treatment of mammalian cells with the per- O-acetylated version of 6AzGlc results in robust labeling of a variety of proteins. Notably, the pattern of this labeling was consistent with O-GlcNAc modifications, suggesting that the enzyme O-GlcNAc transferase is quite promiscuous for its donor sugar substrates. To confirm this possibility, we show that 6AzGlc-treatment results in the labeling of known O-GlcNAcylated proteins, that the UDP-6AzGlc donor sugar is indeed produced in living cells, and that recombinant OGT will accept UDP-6AzGlc as a substrate in vitro. Finally, we use proteomics to first identify several bona fide 6AzGlc-modifications in mammalian cells and then an endogenous O-glucose modification on host cell factor. These results support the conclusion that OGT can endogenously modify proteins with both N-acetyl-glucosamine and glucose, raising the possibility that intracellular O-glucose modification may be a widespread modification under certain conditions or in particular tissues.

Synthesis of UDP-apiose in Bacteria: The marine phototroph Geminicoccus roseus and the plant pathogen Xanthomonas pisi.

The branched-chain sugar apiose was widely assumed to be synthesized only by plant species. In plants, apiose-containing polysaccharides are found in vascularized plant cell walls as the pectic polymers rhamnogalacturonan II and apiogalacturonan. Apiosylated secondary metabolites are also common in many plant species including ancestral avascular bryophytes and green algae. Apiosyl-residues have not been documented in bacteria. In a screen for new bacterial glycan structures, we detected small amounts of apiose in methanolic extracts of the aerobic phototroph Geminicoccus roseus and the pathogenic soil-dwelling bacteria Xanthomonas pisi. Apiose was also present in the cell pellet of X. pisi. Examination of these bacterial genomes uncovered genes with relatively low protein homology to plant UDP-apiose/UDP-xylose synthase (UAS). Phylogenetic analysis revealed that these bacterial UAS-like homologs belong in a clade distinct to UAS and separated from other nucleotide sugar biosynthetic enzymes. Recombinant expression of three bacterial UAS-like proteins demonstrates that they actively convert UDP-glucuronic acid to UDP-apiose and UDP-xylose. Both UDP-apiose and UDP-xylose were detectable in cell cultures of G. roseus and X. pisi. We could not, however, definitively identify the apiosides made by these bacteria, but the detection of apiosides coupled with the in vivo transcription of bUAS and production of UDP-apiose clearly demonstrate that these microbes have evolved the ability to incorporate apiose into glycans during their lifecycles. While this is the first report to describe enzymes for the formation of activated apiose in bacteria, the advantage of synthesizing apiose-containing glycans in bacteria remains unknown. The characteristics of bUAS and its products are discussed.

Chemoenzymatic Synthesis and Applications of Prokaryote-Specific UDP-Sugars.

This method describes the chemoenzymatic synthesis of several nucleotide sugars, which are essential substrates in the biosynthesis of prokaryotic N- and O-linked glycoproteins. Protein glycosylation is now known to be widespread in prokaryotes and proceeds via sequential action of several enzymes, utilizing both common and modified prokaryote-specific sugar nucleotides. The latter, which include UDP-hexoses such as UDP-diNAc-bacillosamine (UDP-diNAcBac), UDP-diNAcAlt, and UDP-2,3-diNAcManA, are also important components of other bacterial and archaeal glycoconjugates. The ready availability of these "high-value" intermediates will enable courses of study into inhibitor screening, glycoconjugate biosynthesis pathway discovery, and unnatural carbohydrate incorporation toward metabolic engineering.

Chemoenzymatic Synthesis of 4-Fluoro-N-Acetylhexosamine Uridine Diphosphate Donors: Chain Terminators in Glycosaminoglycan Synthesis.

Unnatural uridine diphosphate (UDP)-sugar donors, UDP-4-deoxy-4-fluoro-N-acetylglucosamine (4FGlcNAc) and UDP-4-deoxy-4-fluoro-N-acetylgalactosamine (4FGalNAc), were prepared using both chemical and chemoenzymatic syntheses relying on N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). The resulting unnatural UDP-sugar donors were then tested as substrates in glycosaminoglycan synthesis catalyzed by various synthases. UDP-4FGlcNAc was transferred onto an acceptor by Pastuerella multocida heparosan synthase 1 and subsequently served as a chain terminator.

Functional analyses of OcRhS1 and OcUER1 involved in UDP-L-rhamnose biosynthesis in Ornithogalum caudatum.

UDP-L-rhamnose (UDP-Rha) is an important sugar donor for the synthesis of rhamnose-containing compounds in plants. However, only a few enzymes and their encoding genes involved in UDP-Rha biosynthesis are available in plants. Here, two genes encoding rhamnose synthase (RhS) and bi-functional UDP-4-keto-6-deoxy-D-glucose (UDP-4K6DG) 3, 5-epimerase/UDP-4-keto-L-rhamnose (UDP-4KR) 4-keto-reductase (UER) were isolated from Ornithogalum caudatum based on the RNA-Seq data. The OcRhS1 gene has an ORF (open reading frame) of 2019 bp encoding a tri-functional RhS enzyme. In vitro enzymatic assays revealed OcRhS1 can really convert UDP-D-glucose (UDP-Glc) into UDP-Rha via three consecutive reactions. Biochemical evidences indicated that the recombinant OcRhS1 was active in the pH range of 5-11 and over the temperature range of 0-60 °C. The Km value of OcRhS1 for UDP-Glc was determined to be 1.52 × 10-4 M. OcRhS1 is a multi-domain protein with two sets of cofactor-binding motifs. The cofactors dependent properties of OcRhS1 were thus characterized in this research. Moreover, the N-terminal portion of OcRhS1 (OcRhS1-N) was observed to metabolize UDP-Glc to form intermediate UDP-4K6DG. OcUER1 contains an ORF of 906 bp encoding a polypeptide of 301 aa. OcUER1 shared high similarity with the carboxy-terminal domain of OcRhS1 (OcRhS1-C), suggesting its intrinsic ability of converting UDP-4K6DG into UDP-Rha. It was thus reasonably inferred that UDP-Glc could be bio-transformed into UDP-Rha under the collaborating action of OcRhS1-N and OcUER1. The subsequently biochemical assay verified this notion. Importantly, expression profiles of OcRhS1 and OcUER1 revealed their possible involvement in the biosynthesis of rhamnose-containing polysaccharides in O. caudatum.

Functional Characterization of UDP-apiose Synthases from Bryophytes and Green Algae Provides Insight into the Appearance of Apiose-containing Glycans during Plant Evolution.

Apiose is a branched monosaccharide that is present in the cell wall pectic polysaccharides rhamnogalacturonan II and apiogalacturonan and in numerous plant secondary metabolites. These apiose-containing glycans are synthesized using UDP-apiose as the donor. UDP-apiose (UDP-Api) together with UDP-xylose is formed from UDP-glucuronic acid (UDP-GlcA) by UDP-Api synthase (UAS). It was hypothesized that the ability to form Api distinguishes vascular plants from the avascular plants and green algae. UAS from several dicotyledonous plants has been characterized; however, it is not known if avascular plants or green algae produce this enzyme. Here we report the identification and functional characterization of UAS homologs from avascular plants (mosses, liverwort, and hornwort), from streptophyte green algae, and from a monocot (duckweed). The recombinant UAS homologs all form UDP-Api from UDP-glucuronic acid albeit in different amounts. Apiose was detected in aqueous methanolic extracts of these plants. Apiose was detected in duckweed cell walls but not in the walls of the avascular plants and algae. Overexpressing duckweed UAS in the moss Physcomitrella patens led to an increase in the amounts of aqueous methanol-acetonitrile-soluble apiose but did not result in discernible amounts of cell wall-associated apiose. Thus, bryophytes and algae likely lack the glycosyltransferase machinery required to synthesize apiose-containing cell wall glycans. Nevertheless, these plants may have the ability to form apiosylated secondary metabolites. Our data are the first to provide evidence that the ability to form apiose existed prior to the appearance of rhamnogalacturonan II and apiogalacturonan and provide new insights into the evolution of apiose-containing glycans.

Cytomegalovirus-mediated activation of pyrimidine biosynthesis drives UDP-sugar synthesis to support viral protein glycosylation.

Human cytomegalovirus (HCMV) induces numerous changes to the host metabolic network that are critical for high-titer viral replication. We find that HCMV infection substantially induces de novo pyrimidine biosynthetic flux. This activation is important for HCMV replication because inhibition of pyrimidine biosynthetic enzymes substantially decreases the production of infectious virus, which can be rescued through medium supplementation with pyrimidine biosynthetic intermediates. Metabolomic analysis revealed that pyrimidine biosynthetic inhibition considerably reduces the levels of various UDP-sugar metabolites in HCMV-infected, but not mock-infected, cells. Further, UDP-sugar biosynthesis, which provides the sugar substrates required for glycosylation reactions, was found to be induced during HCMV infection. Pyrimidine biosynthetic inhibition also attenuated the glycosylation of the envelope glycoprotein B (gB). Both glycosylation of gB and viral growth were restored by medium supplementation with either UDP-sugar metabolites or pyrimidine precursors. These results indicate that HCMV drives de novo-synthesized pyrimidines to UDP-sugar biosynthesis to support virion protein glycosylation. The importance of this link between pyrimidine biosynthesis and UDP-sugars appears to be partially shared among diverse virus families, because UDP-sugar metabolites rescued the growth attenuation associated with pyrimidine biosynthetic inhibition during influenza A and vesicular stomatitis virus infection, but not murine hepatitis virus infection. In total, our results indicate that viruses can specifically modulate pyrimidine metabolic flux to provide the glycosyl subunits required for protein glycosylation and production of high titers of infectious progeny.

The biosynthesis of UDP-d-FucNAc-4N-(2)-oxoglutarate (UDP-Yelosamine) in Bacillus cereus ATCC 14579: Pat and Pyl, an aminotransferase and an ATP-dependent Grasp protein that ligates 2-oxoglutarate to UDP-4-amino-sugars.

Surface glycan switching is often observed when micro-organisms transition between different biotic and abiotic niches, including biofilms, although the advantages of this switching to the organism are not well understood. Bacillus cereus grown in a biofilm-inducing medium has been shown to synthesize an unusual cell wall polysaccharide composed of the repeating subunit →6)Gal(α1-2)(2-R-hydroxyglutar-5-ylamido)Fuc2NAc4N(α1-6)GlcNAc(ß1→, where galactose is linked to the hydroxyglutarate moiety of FucNAc-4-amido-(2)-hydroxyglutarate. The molecular mechanism involved in attaching 2-hydroxyglutarate to 4-amino-FucNAc has not been determined. Here, we show two genes in B. cereus ATCC 14579 encoding enzymes involved in the synthesis of UDP-FucNAc-4-amido-(2)-oxoglutarate (UDP-Yelosamine), a modified UDP-sugar not previously reported to exist. Using mass spectrometry and real time NMR spectroscopy, we show that Bc5273 encodes a C4″-aminotransferase (herein referred to as Pat) that, in the presence of pyridoxal phosphate, transfers the primary amino group of l-Glu to C-4″ of UDP-4-keto-6-deoxy-d-GlcNAc to form UDP-4-amino-FucNAc and 2-oxoglutarate. Pat also converts 4-keto-xylose, 4-keto-glucose, and 4-keto-2-acetamido-altrose to their corresponding UDP-4-amino-sugars. Bc5272 encodes a carboxylate-amine ligase (herein referred as Pyl) that, in the presence of ATP and Mg(II), adds 2-oxoglutarate to the 4-amino moiety of UDP-4-amino-FucNAc to form UDP-Yelosamine and ADP. Pyl is also able to ligate 2-oxoglutarate to other 4-amino-sugar derivatives to form UDP-Yelose, UDP-Solosamine, and UDP-Aravonose. Characterizing the metabolic pathways involved in the formation of modified nucleotide sugars provides a basis for understanding some of the mechanisms used by bacteria to modify or alter their cell surface polysaccharides in response to changing growth and environmental challenges.

Biochemical analysis and structure determination of bacterial acetyltransferases responsible for the biosynthesis of UDP-N,N'-diacetylbacillosamine.

UDP-N,N'-diacetylbacillosamine (UDP-diNAcBac) is a unique carbohydrate produced by a number of bacterial species and has been implicated in pathogenesis. The terminal step in the formation of this important bacterial sugar is catalyzed by an acetyl-CoA (AcCoA)-dependent acetyltransferase in both N- and O-linked protein glycosylation pathways. This bacterial acetyltransferase is a member of the left-handed ß-helix family and forms a homotrimer as the functional unit. Whereas previous endeavors have focused on the Campylobacter jejuni acetyltransferase (PglD) from the N-linked glycosylation pathway, structural characterization of the homologous enzymes in the O-linked glycosylation pathways is lacking. Herein, we present the apo-crystal structures of the acetyltransferase domain (ATD) from the bifunctional enzyme PglB (Neisseria gonorrhoeae) and the full-length acetyltransferase WeeI (Acinetobacter baumannii). Additionally, a PglB-ATD structure was solved in complex with AcCoA. Surprisingly, this structure reveals a contrasting binding mechanism for this substrate when compared with the AcCoA-bound PglD structure. A comparison between these findings and the previously solved PglD crystal structures illustrates a dichotomy among N- and O-linked glycosylation pathway enzymes. Based upon these structures, key residues in the UDP-4-amino and AcCoA binding pockets were mutated to determine their effect on binding and catalysis in PglD, PglB-ATD, and WeeI. Last, a phylogenetic analysis of the aforementioned acetyltransferases was employed to illuminate the diversity among N- and O-linked glycosylation pathway enzymes.

Biosynthesis of nucleotide sugars by a promiscuous UDP-sugar pyrophosphorylase from Arabidopsis thaliana (AtUSP).

Nucleotide sugars are activated forms of monosaccharides and key intermediates of carbohydrate metabolism in all organisms. The availability of structurally diverse nucleotide sugars is particularly important for the characterization of glycosyltransferases. Given that limited methods are available for preparation of nucleotide sugars, especially their useful non-natural derivatives, we introduced herein an efficient one-step three-enzyme catalytic system for the synthesis of nucleotide sugars from monosaccharides. In this study, a promiscuous UDP-sugar pyrophosphorylase (USP) from Arabidopsis thaliana (AtUSP) was used with a galactokinase from Streptococcus pneumoniae TIGR4 (SpGalK) and an inorganic pyrophosphatase (PPase) to effectively synthesize four UDP-sugars. AtUSP has better tolerance for C4-derivatives of Gal-1-P compared to UDP-glucose pyrophosphorylase from S. pneumoniae TIGR4 (SpGalU). Besides, the nucleotide substrate specificity and kinetic parameters of AtUSP were systematically studied. AtUSP exhibited considerable activity toward UTP, dUTP and dTTP, the yield of which was 87%, 85% and 84%, respectively. These results provide abundant information for better understanding of the relationship between substrate specificity and structural features of AtUSP.

Human UDP-α-D-xylose synthase and Escherichia coli ArnA conserve a conformational shunt that controls whether xylose or 4-keto-xylose is produced.

Human UDP-α-D-xylose synthase (hUXS) is a member of the short-chain dehydrogenase/reductase family of nucleotide-sugar modifying enzymes. hUXS contains a bound NAD(+) cofactor that it recycles by first oxidizing UDP-α-D-glucuronic acid (UGA), and then reducing the UDP-α-D-4-keto-xylose (UX4O) to produce UDP-α-D-xylose (UDX). Despite the observation that purified hUXS contains a bound cofactor, it has been reported that exogenous NAD(+) will stimulate enzyme activity. Here we show that a small fraction of hUXS releases the NADH and UX4O intermediates as products during turnover. The resulting apoenzyme can be rescued by exogenous NAD(+), explaining the apparent stimulatory effect of added cofactor. The slow release of NADH and UX4O as side products by hUXS is reminiscent of the Escherichia coli UGA decarboxylase (ArnA), a related enzyme that produces NADH and UX4O as products. We report that ArnA can rebind NADH and UX4O to slowly make UDX. This means that both enzymes share the same catalytic machinery, but differ in the preferred final product. We present a bifurcated rate equation that explains how the substrate is shunted to the distinct final products. Using a new crystal structure of hUXS, we identify the structural elements of the shunt and propose that the local unfolding of the active site directs reactants toward the preferred products. Finally, we present evidence that the release of NADH and UX4O involves a cooperative conformational change that is conserved in both enzymes.

Efficient one-pot multienzyme synthesis of UDP-sugars using a promiscuous UDP-sugar pyrophosphorylase from Bifidobacterium longum (BLUSP).

A promiscuous UDP-sugar pyrophosphorylase (BLUSP) was cloned from Bifidobacterium longum strain ATCC55813 and used efficiently with a Pasteurella multocida inorganic pyrophosphatase (PmPpA) with or without a monosaccharide 1-kinase for one-pot multienzyme synthesis of UDP-galactose, UDP-glucose, UDP-mannose, and their derivatives. Further chemical diversification of a UDP-mannose derivative resulted in the formation of UDP-N-acetylmannosamine.

Biosynthesis of UDP-4-keto-6-deoxyglucose and UDP-rhamnose in pathogenic fungi Magnaporthe grisea and Botryotinia fuckeliana.

There is increasing evidence that in several fungi, rhamnose-containing glycans are involved in processes that affect host-pathogen interactions, including adhesion, recognition, virulence, and biofilm formation. Nevertheless, little is known about the pathways for the synthesis of these glycans. We show that rhamnose is present in glycans isolated from the rice pathogen Magnaporthe grisea and from the plant pathogen Botryotinia fuckeliana. We also provide evidence that these fungi produce UDP-rhamnose. This is in contrast to bacteria where dTDP-rhamnose is the activated form of this sugar. In bacteria, formation of dTDP-rhamnose requires three enzymes. Here, we demonstrate that in fungi only two genes are required for UDP-Rha synthesis. The first gene encodes a UDP-glucose-4,6-dehydratase that converts UDP-glucose to UDP-4-keto-6-deoxyglucose. The product was shown by time-resolved (1)H NMR spectroscopy to exist in solution predominantly as a hydrated form along with minor amounts of a keto form. The second gene encodes a bifunctional UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase that converts UDP-4-keto-6-deoxyglucose to UDP-rhamnose. Sugar composition analysis and gene expression studies at different stages of growth indicate that the synthesis of rhamnose-containing glycans is under tissue-specific regulation. Together, our results provide new insight into the formation of rhamnose-containing glycans during the fungal life cycle. The role of these glycans in the interactions between fungal pathogens and their hosts is discussed. Knowledge of the metabolic pathways involved in the formation of rhamnose-containing glycans may facilitate the development of drugs to combat fungal diseases in humans, as to the best of our knowledge mammals do not make these types of glycans.

A common structural blueprint for plant UDP-sugar-producing pyrophosphorylases.

Plant pyrophosphorylases that are capable of producing UDP-sugars, key precursors for glycosylation reactions, include UDP-glucose pyrophosphorylases (A- and B-type), UDP-sugar pyrophosphorylase and UDP-N-acetylglucosamine pyrophosphorylase. Although not sharing significant homology at the amino acid sequence level, the proteins share a common structural blueprint. Their structures are characterized by the presence of the Rossmann fold in the central (catalytic) domain linked to enzyme-specific N-terminal and C-terminal domains, which may play regulatory functions. Molecular mobility between these domains plays an important role in substrate binding and catalysis. Evolutionary relationships and the role of (de)oligomerization as a regulatory mechanism are discussed.

N-acetylglucosamine 6-phosphate deacetylase (nagA) is required for N-acetyl glucosamine assimilation in Gluconacetobacter xylinus.

Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tet(r); named as ΔnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species.