Cytosolic UDP-L-arabinose synthesis by bifunctional UDP-glucose 4-epimerases in Arabidopsis.

L-Arabinose (L-Ara) is a plant-specific sugar found in cell wall polysaccharides, proteoglycans, glycoproteins, and small glycoconjugates, which play physiologically important roles in cell proliferation and other essential cellular processes. L-Ara is synthesized as UDP-L-arabinose (UDP-L-Ara) from UDP-xylose (UDP-Xyl) by UDP-Xyl 4-epimerases (UXEs), a type of de novo synthesis of L-Ara unique to plants. In Arabidopsis, the Golgi-localized UXE AtMUR4 is the main contributor to UDP-L-Ara synthesis. However, cytosolic bifunctional UDP-glucose 4-epimerases (UGEs) with UXE activity, AtUGE1, and AtUGE3 also catalyze this reaction. For the present study, we first examined the physiological importance of bifunctional UGEs in Arabidopsis. The uge1 and uge3 mutants enhanced the dwarf phenotype of mur4 and further reduced the L-Ara content in cell walls, suggesting that bifunctional UGEs contribute to UDP-L-Ara synthesis. Through the introduction of point mutations exchanging corresponding amino acid residues between AtUGE1 with high UXE activity and AtUGE2 with low UXE activity, two mutations that increase relative UXE activity of AtUGE2 were identified. The crystal structures of AtUGE2 in complex forms with NAD+ and NAD+/UDP revealed that the UDP-binding domain of AtUGE2 has a more closed conformation and smaller sugar-binding site than bacterial and mammalian UGEs, suggesting that plant UGEs have the appropriate size and shape for binding UDP-Xyl and UDP-L-Ara to exhibit UXE activity. The presented results suggest that the capacity for cytosolic synthesis of UDP-L-Ara was acquired by the small sugar-binding site and several mutations of UGEs, enabling diversified utilization of L-Ara in seed plants.

The galactose metabolism genes UGE1 and UGM1 are novel virulence factors of the maize anthracnose fungus Colletotrichum graminicola.

Fungal cell walls represent the frontline contact with the host and play a prime role in pathogenesis. While the roles of the cell wall polymers like chitin and branched ß-glucan are well understood in vegetative and pathogenic development, that of the most prominent galactose-containing polymers galactosaminogalactan and fungal-type galactomannan is unknown in plant pathogenic fungi. Mining the genome of the maize pathogen Colletotrichum graminicola identified the single-copy key galactose metabolism genes UGE1 and UGM1, encoding a UDP-glucose-4-epimerase and UDP-galactopyranose mutase, respectively. UGE1 is thought to be required for biosynthesis of both polymers, whereas UGM1 is specifically required for fungal-type galactomannan formation. Promoter:eGFP fusion strains revealed that both genes are expressed in vegetative and in pathogenic hyphae at all stages of pathogenesis. Targeted deletion of UGE1 and UGM1, and fluorescence-labeling of galactosaminogalactan and fungal-type galactomannan confirmed that Δuge1 mutants were unable to synthesize either of these polymers, and Δugm1 mutants did not exhibit fungal-type galactomannan. Appressoria of Δuge1, but not of Δugm1 mutants, were defective in adhesion, highlighting a function of galactosaminogalactan in the establishment of these infection cells on hydrophobic surfaces. Both Δuge1 and Δugm1 mutants showed cell wall defects in older vegetative hyphae and severely reduced appressorial penetration competence. On intact leaves of Zea mays, both mutants showed strongly reduced disease symptom severity, indicating that UGE1 and UGM1 represent novel virulence factors of C. graminicola.

Novel variants in GALE cause syndromic macrothrombocytopenia by disrupting glycosylation and thrombopoiesis.

Glycosylation is recognized as a key process for proper megakaryopoiesis and platelet formation. The enzyme uridine diphosphate (UDP)-galactose-4-epimerase, encoded by GALE, is involved in galactose metabolism and protein glycosylation. Here, we studied 3 patients from 2 unrelated families who showed lifelong severe thrombocytopenia, bleeding diathesis, mental retardation, mitral valve prolapse, and jaundice. Whole-exome sequencing revealed 4 variants that affect GALE, 3 of those previously unreported (Pedigree A, p.Lys78ValfsX32 and p.Thr150Met; Pedigree B, p.Val128Met; and p.Leu223Pro). Platelet phenotype analysis showed giant and/or grey platelets, impaired platelet aggregation, and severely reduced alpha and dense granule secretion. Enzymatic activity of the UDP-galactose-4-epimerase enzyme was severely decreased in all patients. Immunoblotting of platelet lysates revealed reduced GALE protein levels, a significant decrease in N-acetyl-lactosamine (LacNAc), showing a hypoglycosylation pattern, reduced surface expression of gylcoprotein Ibα-IX-V (GPIbα-IX-V) complex and mature ß1 integrin, and increased apoptosis. In vitro studies performed with patients-derived megakaryocytes showed normal ploidy and maturation but decreased proplatelet formation because of the impaired glycosylation of the GPIbα and ß1 integrin, and reduced externalization to megakaryocyte and platelet membranes. Altered distribution of filamin A and actin and delocalization of the von Willebrand factor were also shown. Overall, this study expands our knowledge of GALE-related thrombocytopenia and emphasizes the critical role of GALE in the physiological glycosylation of key proteins involved in platelet production and function.

Overexpression of UDP-sugar pyrophosphorylase leads to higher sensitivity towards galactose, providing new insights into the mechanisms of galactose toxicity in plants.

Galactose toxicity (Gal-Tox) is a widespread phenomenon ranging from Escherichia coli to mammals and plants. In plants, the predominant pathway for the conversion of galactose into UDP-galactose (UDP-Gal) and UDP-glucose is catalyzed by the enzymes galactokinase, UDP-sugar pyrophosphorylase (USP) and UDP-galactose 4-epimerase. Galactose is a major component of cell wall polymers, glycolipids and glycoproteins; therefore, it becomes surprising that exogenous addition of galactose leads to drastic root phenotypes including cessation of primary root growth and induction of lateral root formation. Currently, little is known about galactose-mediated toxicity in plants. In this study, we investigated the role of galactose-containing metabolites like galactose-1-phosphate (Gal-1P) and UDP-Gal in Gal-Tox. Recently published data from mouse models suggest that a reduction of the Gal-1P level via an mRNA-based therapy helps to overcome Gal-Tox. To test this hypothesis in plants, we created Arabidopsis thaliana lines overexpressing USP from Pisum sativum. USP enzyme assays confirmed a threefold higher enzyme activity in the overexpression lines leading to a significant reduction of the Gal-1P level in roots. Interestingly, the overexpression lines are phenotypically more sensitive to the exogenous addition of galactose (0.5 mmol L-1 Gal). Nucleotide sugar analysis via high-performance liquid chromatography-mass spectrometry revealed highly elevated UDP-Gal levels in roots of seedlings grown on 1.5 mmol L-1 galactose versus 1.5 mmol L-1 sucrose. Analysis of plant cell wall glycans by comprehensive microarray polymer profiling showed a high abundance of antibody binding recognizing arabinogalactanproteins and extensins under Gal-feeding conditions, indicating that glycoproteins are a major target for elevated UDP-Gal levels in plants.

A new function of a putative UDP-glucose 4-epimerase on the expression of glycoside hydrolase genes in Aspergillus aculeatus.

In order to figure out the induction mechanisms of glycoside hydrolase genes in Aspergillus aculeatus, we screened approximately 9,000 transfer DNA (T-DNA)-inserted mutants for positive regulators involved in the induction. Since the mutants possess the orotidine 5'-monophosphate decarboxylase gene as a reporter gene to monitor the cellulose-responsive expression of the cellobiohydrolase I gene (cbhI), candidate strains were isolated by counterselection against 5-fluoroorotic acid (5-FOA). One 5-FOA-resistant mutant harboring the T-DNA at the uge5 locus showed reduced cellulose utilization and cbhI expression. A. aculeatus Uge5 is homologous to Aspergillus fumigatus uge5 (Afu5g10780; E-value, 0.0; identities, 93%), which catalyzes the conversion of uridine diphosphate (UDP)-glucose to UDP-galactopyranose. The uge5 deletion mutant in A. aculeatus (Δuge5) showed reduced conidium formation on minimal media supplemented with galactose, locust bean gum (LBG), and guar gum as a carbon source. ß-1,4-Endoglucanase and ß-1,4-mannanase production in submerged culture containing LBG was reduced to 10% and 6% of the control strain at day 5, respectively, but no difference was observed in cultures containing wheat bran. The expression of major cellulolytic and mannolytic genes in the presence of mannobiose in Δuge5 was reduced to less than 15% of the control strain, while cellobiose-responsive expression was only modestly reduced at early inducing time points. Since all test genes were controlled by a transcription factor ManR, these data demonstrate that Uge5 is involved in inducer-dependent selective expression of genes controlled via ManR. KEY POINTS: • UDP-glucose 4-epimerase (Uge5) regulates expression of glycosyl hydrolase genes. • ManR regulates both cellobiose- and mannobiose-responsive expression. • Uge5 plays a key role in mannobiose-responsive expression.

LcERF2 modulates cell wall metabolism by directly targeting a UDP-glucose-4-epimerase gene to regulate pedicel development and fruit abscission of litchi.

Elucidating the biochemical and molecular basis of premature abscission in fruit crops should help develop strategies to enhance fruit set and yield. Here, we report that LcERF2 contributes to differential abscission rates and responses to ethylene in Litchi chinensis (litchi). Reduced LcERF2 expression in litchi was observed to reduce fruit abscission, concurrent with enhanced pedicel growth and increased levels of hexoses, particularly galactose, as well as pectin abundance in the cell wall. Ecoptic expression of LcERF2 in Arabidopsis thaliana caused enhanced petal abscission, together with retarded plant growth and reduced pedicel galactose and pectin contents. Transcriptome analysis indicated that LcERF2 modulates the expression of genes involved in cell wall modification. Yeast one-hybrid, dual-luciferase reporter and electrophoretic mobility shift assays all demonstrated that a UDP-glucose-4-epimerase gene (LcUGE) was the direct downstream target of LcERF2. This result was further supported by a significant reduction in the expression of the A. thaliana homolog AtUGE2-4 in response to LcERF2 overexpression. Significantly reduced pedicel diameter and enhanced litchi fruit abscission were observed in response to LcUGE silencing. We conclude that LcERF2 mediates fruit abscission by orchestrating cell wall metabolism, and thus pedicel growth, in part by repressing the expression of LcUGE.

Disruption of OsPHD1, Encoding a UDP-Glucose Epimerase, Causes JA Accumulation and Enhanced Bacterial Blight Resistance in Rice.

Lesion mimic mutants (LMMs) have been widely used in experiments in recent years for studying plant physiological mechanisms underlying programmed cell death (PCD) and defense responses. Here, we identified a lesion mimic mutant, lm212-1, which cloned the causal gene by a map-based cloning strategy, and verified this by complementation. The causal gene, OsPHD1, encodes a UDP-glucose epimerase (UGE), and the OsPHD1 was located in the chloroplast. OsPHD1 was constitutively expressed in all organs, with higher expression in leaves and other green tissues. lm212-1 exhibited decreased chlorophyll content, and the chloroplast structure was destroyed. Histochemistry results indicated that H2O2 is highly accumulated and cell death is occurred around the lesions in lm212-1. Compared to the wild type, expression levels of defense-related genes were up-regulated, and resistance to bacterial pathogens Xanthomonas oryzae pv. oryzae (Xoo) was enhanced, indicating that the defense response was activated in lm212-1, ROS production was induced by flg22, and chitin treatment also showed the same result. Jasmonic acid (JA) and methyl jasmonate (MeJA) increased, and the JA signaling pathways appeared to be disordered in lm212-1. Additionally, the overexpression lines showed the same phenotype as the wild type. Overall, our findings demonstrate that OsPHD1 is involved in the regulation of PCD and defense response in rice.

Human UDP-galactose 4'-epimerase (GALE) is required for cell-surface glycome structure and function.

Glycan biosynthesis relies on nucleotide sugars (NSs), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo, signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease. However, how mammalian cells regulate NS levels and pathway flux remains largely uncharacterized. To address this knowledge gap, here we examined UDP-galactose 4'-epimerase (GALE), which interconverts two pairs of essential NSs. Using immunoblotting, flow cytometry, and LC-MS-based glycolipid and glycan profiling, we found that CRISPR/Cas9-mediated GALE deletion in human cells triggers major imbalances in NSs and dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the cell-surface death receptor FS-7-associated surface antigen. In particular, we observed substantial decreases in total sialic acid, galactose, and GalNAc levels in glycans. These changes also directly impacted cell signaling, as GALE-/- cells exhibited FS-7-associated surface antigen ligand-induced apoptosis. Our results reveal a role of GALE-mediated NS regulation in death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome.

Inherited thrombocytopenia associated with mutation of UDP-galactose-4-epimerase (GALE).

Severe thrombocytopenia, characterized by dysplastic megakaryocytes and intracranial bleeding, was diagnosed in six individuals from a consanguineous kindred. Three of the individuals were successfully treated by bone marrow transplant. Whole-exome sequencing and homozygosity mapping of multiple family members, coupled with whole-genome sequencing to reveal shared non-coding variants, revealed one potentially functional variant segregating with thrombocytopenia under a recessive model: GALE p.R51W (c.C151T, NM_001127621). The mutation is extremely rare (allele frequency = 2.5 × 10-05), and the likelihood of the observed co-segregation occurring by chance is 1.2 × 10-06. GALE encodes UDP-galactose-4-epimerase, an enzyme of galactose metabolism and glycosylation responsible for two reversible reactions: interconversion of UDP-galactose with UDP-glucose and interconversion of UDP-N-acetylgalactosamine with UDP-N-acetylglucosamine. The mutation alters an amino acid residue that is conserved from yeast to humans. The variant protein has both significantly lower enzymatic activity for both interconversion reactions and highly significant thermal instability. Proper glycosylation is critical to normal hematopoiesis, in particular to megakaryocyte and platelet development, as reflected in the presence of thrombocytopenia in the context of congenital disorders of glycosylation. Mutations in GALE have not previously been associated with thrombocytopenia. Our results suggest that GALE p.R51W is inadequate for normal glycosylation and thereby may impair megakaryocyte and platelet development. If other mutations in GALE are shown to have similar consequences, this gene may be proven to play a critical role in hematopoiesis.

ARPI, ß-AS, and UGE regulate glycyrrhizin biosynthesis in Glycyrrhiza uralensis hairy roots.

KEY MESSAGE: ARPI, ß-AS, and UGE were cloned from G. uralensis and their regulatory effects on glycyrrhizin biosynthesis were investigated. ß-AS and UGE but not ARPI positively regulate the biosynthesis of glycyrrhizin. Glycyrrhiza uralensis Fisch. has been used to treat respiratory, gastric, and liver diseases since ancient China. The most important and widely studied active component in G. uralensis is glycyrrhizin (GC). Our pervious RNA-Seq study shows that GC biosynthesis is regulated by multiple biosynthetic pathways. In this study, three target genes, ARPI, ß-AS, and UGE from different pathways were selected and their regulatory effects on GC biosynthesis were investigated using G. uralensis hairy roots. Our data show that hairy roots knocking out ARPI or UGE died soon after induction, indicating that the genes are essential for the growth of G. uralensis hairy roots. Hairy roots with ß-AS knocked out grew healthily. However, they failed to produce GC, suggesting that ß-AS is required for triterpenoid skeleton formation. Conversely, overexpression of UGE or ß-AS significantly increased the GC content, whereas overexpression of ARPI had no obvious effects on GC accumulation in G. uralensis hairy roots. Our findings demonstrate that ß-AS and UGE positively regulate the biosynthesis of GC.

BRITTLE PLANT1 is required for normal cell wall composition and mechanical strength in rice.

A series of nucleotide sugar interconversion enzymes (NSEs) generate the activated sugar donors required for biosynthesis of cell wall matrix polysaccharides and glycoproteins. UDP-glucose 4-epimerases (UGEs) are NSEs that function in the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal). The roles of UDP-glucose 4-epimerases in monocots remain unclear due to redundancy in the pathways. Here, we report a brittle plant (bp1) rice mutant that exhibits brittle leaves and culms at all growth stages. The mutant culms had reduced levels of rhamnogalacturonan I, homogalacturonan, and arabinogalactan proteins. Moreover, the mutant had altered contents of uronic acids, neutral noncellulosic monosaccharides, and cellulose. Map-based cloning demonstrated that OsBP1 encodes a UDP-glucose 4-epimerase (OsUGE2), a cytosolic protein. We also show that BP1 can form homo- and hetero-protein complexes with other UGE family members and with UDP-galactose transporters 2 (OsUGT2) and 3 (OsUGT3), which may facilitate the channeling of Gal to polysaccharides and proteoglycans. Our results demonstrate that BP1 participates in regulating the sugar composition and structure of rice cell walls.

Structure of an antibiotic-synthesizing UDP-glucuronate 4-epimerase MoeE5 in complex with substrate.

The epimerase MoeE5 from Streptomyces viridosporus converts UDP-glucuronic acid (UDP-GlcA) to UDP-galacturonic acid (UDP-GalA) to provide the first sugar in synthesizing moenomycin, a potent inhibitor against bacterial peptidoglycan glycosyltransferases. The enzyme belongs to the UDP-hexose 4-epimerase family, and uses NAD+ as its cofactor. Here we present the complex crystal structures of MoeE5/NAD+/UDP-GlcA and MoeE5/NAD+/UDP-glucose, determined at 1.48 Šand 1.66 Šresolution. The cofactor NAD+ is bound to the N-terminal Rossmann-fold domain and the substrate is bound to the smaller C-terminal domain. In both crystals the C4 atom of the sugar moiety of the substrate is in close proximity to the C4 atom of the nicotinamide of NAD+, and the O4 atom of the sugar is also hydrogen bonded to the side chain of Tyr154, suggesting a productive binding mode. As the first complex structure of this protein family with a bound UDP-GlcA in the active site, it shows an extensive hydrogen-bond network between the enzyme and the substrate. We further built a model with the product UDP-GalA, and found that the unique Arg192 of MoeE5 might play an important role in the catalytic pathway. Consequently, MoeE5 is likely a specific epimerase for UDP-GlcA to UDP-GalA conversion, rather than a promiscuous enzyme as some other family members.

A novel rice fragile culm 24 mutant encodes a UDP-glucose epimerase that affects cell wall properties and photosynthesis.

UDP-glucose epimerases (UGEs) are essential enzymes for catalysing the conversion of UDP-glucose (UDP-Glc) into UDP-galactose (UDP-Gal). Although UDP-Gal has been well studied as the substrate for the biosynthesis of carbohydrates, glycolipids, and glycoproteins, much remains unknown about the biological function of UGEs in plants. In this study, we selected a novel rice fragile culm 24 (Osfc24) mutant and identified it as a nonsense mutation of the FC24/OsUGE2 gene. The Osfc24 mutant shows a brittleness phenotype with significantly altered cell wall composition and disrupted orientation of the cellulose microfibrils. We found significantly reduced accumulation of arabinogalactan proteins in the cell walls of the mutant, which may consequently affect plant growth and cell wall deposition, and be responsible for the altered cellulose microfibril orientation. The mutant exhibits dwarfism and paler leaves with significantly decreased contents of galactolipids and chlorophyll, resulting in defects in plant photosynthesis. Based on our results, we propose a model for how OsUGE2 participates in two distinct metabolic pathways to co-modulate cellulose biosynthesis and cell wall assembly by dynamically providing UDP-Gal and UDP-Glc substrates.

Pathophysiology and targets for treatment in hereditary galactosemia: A systematic review of animal and cellular models.

Since the first description of galactosemia in 1908 and despite decades of research, the pathophysiology is complex and not yet fully elucidated. Galactosemia is an inborn error of carbohydrate metabolism caused by deficient activity of any of the galactose metabolising enzymes. The current standard of care, a galactose-restricted diet, fails to prevent long-term complications. Studies in cellular and animal models in the past decades have led to an enormous progress and advancement of knowledge. Summarising current evidence in the pathophysiology underlying hereditary galactosemia may contribute to the identification of treatment targets for alternative therapies that may successfully prevent long-term complications. A systematic review of cellular and animal studies reporting on disease complications (clinical signs and/or biochemical findings) and/or treatment targets in hereditary galactosemia was performed. PubMed/MEDLINE, EMBASE, and Web of Science were searched, 46 original articles were included. Results revealed that Gal-1-P is not the sole pathophysiological agent responsible for the phenotype observed in galactosemia. Other currently described contributing factors include accumulation of galactose metabolites, uridine diphosphate (UDP)-hexose alterations and subsequent impaired glycosylation, endoplasmic reticulum (ER) stress, altered signalling pathways, and oxidative stress. galactokinase (GALK) inhibitors, UDP-glucose pyrophosphorylase (UGP) up-regulation, uridine supplementation, ER stress reducers, antioxidants and pharmacological chaperones have been studied, showing rescue of biochemical and/or clinical symptoms in galactosemia. Promising co-adjuvant therapies include antioxidant therapy and UGP up-regulation. This systematic review provides an overview of the scattered information resulting from animal and cellular studies performed in the past decades, summarising the complex pathophysiological mechanisms underlying hereditary galactosemia and providing insights on potential treatment targets.

Deciphering the Biosynthesis of TDP-ß-l-oleandrose in Avermectin.

Avermectin (AVM) refers to eight macrolides containing a common l-oleandrosyl disaccharide chain indispensable to their antiparasitic bioactivities. We delineated the biosynthetic pathway of TDP-ß-l-oleandrose (1), the sugar donor of AVM, by characterizing AveBVIII, AveBV, and AveBVII as TDP-sugar 3-ketoreductase, 5-epimerase, and 3-O-methyltransferase, respectively. On the basis of this pathway, we successfully reconstituted the biosynthesis of 1 in Escherichia coli. Our work completes the biosynthetic pathway of AVM and lays a solid foundation for further studies.

l-Arabinose induces d-galactose catabolism via the Leloir pathway in Aspergillus nidulans.

l-Arabinose and d-galactose are the principal constituents of l-arabinogalactan, and also co-occur in other hemicelluloses and pectins. In this work we hypothesized that similar to the induction of relevant glycoside hydrolases by monomers liberated from these plant heteropolymers, their respective catabolisms in saprophytic and phytopathogenic fungi may respond to the presence of the other sugar to promote synergistic use of the complex growth substrate. We showed that these two sugars are indeed consumed simultaneously by Aspergillus nidulans, while l-arabinose is utilised faster in the presence than in the absence of d-galactose. Furthermore, the first two genes of the Leloir pathway for d-galactose catabolism - encoding d-galactose 1-epimerase and galactokinase - are induced more rapidly by l-arabinose than by d-galactose eventhough deletion mutants thereof grow as well as a wild type strain on the pentose. d-Galactose 1-epimerase is hyperinduced by l-arabinose, d-xylose and l-arabitol but not by xylitol. The results suggest that in A. nidulans, l-arabinose and d-xylose - both requiring NADPH for their catabolisation - actively promote the enzyme infrastructure necessary to convert ß-d-galactopyranose via the Leloir pathway with its α-anomer specific enzymes, into ß-d-glucose-6-phosphate (the starting substrate of the oxidative part of the pentose phosphate pathway) even in the absence of d-galactose.

The evolution of a Web resource: The Galactosemia Proteins Database 2.0.

Galactosemia Proteins Database 2.0 is a Web-accessible resource collecting information about the structural and functional effects of the known variations associated to the three different enzymes of the Leloir pathway encoded by the genes GALT, GALE, and GALK1 and involved in the different forms of the genetic disease globally called "galactosemia." It represents an evolution of two available online resources we previously developed, with new data deriving from new structures, new analysis tools, and new interfaces and filters in order to improve the quality and quantity of information available for different categories of users. We propose this new resource both as a landmark for the entire world community of galactosemia and as a model for the development of similar tools for other proteins object of variations and involved in human diseases.

KfoA, the UDP-glucose-4-epimerase of Escherichia coli strain O5:K4:H4, shows preference for acetylated substrates.

Capsule of Escherichia coli O5:K4:H4 is formed of a chondroitin-repeat disaccharide unit of glucuronic acid (GlcA)-N-acetylgalactosamine (GalNAc). This polysaccharide, commonly referred to as K4CP, is a potentially important source of precursors for chemoenzymatic or bioengineering synthesis of chondroitin sulfate. KfoA, encoded by a gene from region 2 of the K4 capsular gene cluster, shows high homology to the UDP-glucose-4-epimerase (GalE) from E. coli. KfoA is reputed to be responsible for uridine 5'-diphosphate-N-acetylgalactosamine (UDP-GalNAc) supply for K4CP biosynthesis in vivo, but it has not been biochemically characterized. Here, we probed the substrate specificity of KfoA by a capillary electrophoresis (CE)-based method. KfoA could epimerize both acetylated and non-acetylated substrates, but its k cat/K m value for UDP-GlcNAc was approximately 1300-fold that for UDP-Glc. Recombinant KfoA showed a strong preference for acetylated substrates in vitro. The conclusion that KfoA is a higher efficiency UDP-GalNAc provider than GalE was supported by a coupled assay developed based on the donor-acceptor combination specificity of E. coli K4 chondroitin polymerase (KfoC). Furthermore, residue Ser-301, located near the UDP-GlcNAc binding pocket, plays an important role in the determination of the conversion ratio of UDP-GlcNAc to UDP-GalNAc by KfoA. Our results deepen the understanding of the mechanism of KfoA and will assist in the research into the metabolic engineering for chondroitin sulfate production.

Characterization and Mutational Analysis of Two UDP-Galactose 4-Epimerases in Streptococcus pneumoniae TIGR4.

Current clinical treatments for pneumococcal infections have many limitations and are faced with many challenges. New capsular polysaccharide structures must be explored to cope with diseases caused by different serotypes of Streptococcus pneumoniae. UDP-galactose 4-epimerase (GalE) is an essential enzyme involved in polysaccharide synthesis. It is an important virulence factor in many bacterial pathogens. In this study, we found that two genes (galEsp1 and galEsp2) are responsible for galactose metabolism in pathogenic S. pneumoniae TIGR4. Both GalESp1 and GalESp2 were shown to catalyze the epimerization of UDP-glucose (UDP-Glc)/UDP-galactose (UDP-Gal), but only GalESp2 was shown to catalyze the epimerization of UDP-N-acetylglucosamine (UDP-GlcNAc)/UDP-N-acetylgalactosamine (UDP-GalNAc). Interestingly, GalESp2 had 3-fold higher epimerase activity toward UDP-Glc/UDP-Gal than GalESp1. The biochemical properties of GalESp2 were studied. GalESp2 was stable over a wide range of temperatures, between 30 and 70°C, at pH 8.0. The K86G substitution caused GalESp2 to lose its epimerase activity toward UDP-Glc and UDP-Gal; however, substitution C300Y in GalESp2 resulted in only decreased activity toward UDP-GlcNAc and UDP-GalNAc. These results indicate that the Lys86 residue plays a critical role in the activity and substrate specificity of GalESp2.

UDP-Glucose 4-Epimerase and ß-1,4-Galactosyltransferase from the Oyster Magallana gigas as Valuable Biocatalysts for the Production of Galactosylated Products.

Uridine diphosphate galactose (UDP-galactose) is a valuable building block in the enzymatic synthesis of galactose-containing glycoconjugates. UDP-glucose 4-epimerase (UGE) is an enzyme which catalyzes the reversible conversion of abundantly available UDP-glucose to UDP-galactose. Herein, we described the cloning, expression, purification, and biochemical characterization of an unstudied UGE from the oyster Magallana gigas (MgUGE). Activity tests of recombinantly expressed MgUGE, using HPLC (high-performance liquid chromatography), mass spectrometry, and photometric assays, showed an optimal temperature of 16 °C, and reasonable thermal stability up to 37 °C. No metal ions were required for enzymatic activity. The simple nickel-affinity-purification procedure makes MgUGE a valuable biocatalyst for the synthesis of UDP-galactose from UDP-glucose. The biosynthetic potential of MgUGE was further exemplified in a coupled enzymatic reaction with an oyster-derived ß-1,4-galactosyltransferase (MgGalT7), allowing the galactosylation of the model substrate para-nitrophenol xylose (pNP-xylose) using UDP-glucose as the starting material.