Selective analysis of intracellular UDP-GlcNAc and UDP-GalNAc by hydrophilic interaction liquid chromatography-mass spectrometry.

Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) is one of the major nucleotide sugars in living organisms and serves as the key donor substrate for the post-translational modification of protein O-GlcNAcylation. It undergoes interconversion to its epimer uridine diphosphate-N-acetylgalactosamine (UDP-GalNAc), which acts as a sugar donor initiating mucin-type O-linked glycosylation. The intracellular levels of the two differ between the cell lines and largely fluctuate in response to metabolic perturbations, and recent studies have focused on the details of their biosynthesis or turnover. However, due to their similar chemical properties, sufficient resolution for the two epimers required non-volatile mobile phases that cannot be applied directly to a mass spectrometer. In this study, to implement simple liquid chromatography-mass spectrometry for UDP-GlcNAc and UDP-GalNAc, we optimized a condition of hydrophilic interaction liquid chromatography-mass spectrometry. We found that the use of ammonium hydroxide and an amide column with an optimized water-acetonitrile ratio, flow rate, and column temperature, provided complete separation of the two. The method allowed the analysis of intracellular levels, a stable isotope-labeled target, and patterns of product ion spectra in a single run with fewer sample preparation steps. The new method can be widely used for mass spectrometric analysis of UDP-GlcNAc and UDP-GalNAc.

Structural basis for directional chitin biosynthesis.

Chitin, the most abundant aminopolysaccharide in nature, is an extracellular polymer consisting of N-acetylglucosamine (GlcNAc) units1. The key reactions of chitin biosynthesis are catalysed by chitin synthase2-4, a membrane-integrated glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to a growing chitin chain. However, the precise mechanism of this process has yet to be elucidated. Here we report five cryo-electron microscopy structures of a chitin synthase from the devastating soybean root rot pathogenic oomycete Phytophthora sojae (PsChs1). They represent the apo, GlcNAc-bound, nascent chitin oligomer-bound, UDP-bound (post-synthesis) and chitin synthase inhibitor nikkomycin Z-bound states of the enzyme, providing detailed views into the multiple steps of chitin biosynthesis and its competitive inhibition. The structures reveal the chitin synthesis reaction chamber that has the substrate-binding site, the catalytic centre and the entrance to the polymer-translocating channel that allows the product polymer to be discharged. This arrangement reflects consecutive key events in chitin biosynthesis from UDP-GlcNAc binding and polymer elongation to the release of the product. We identified a swinging loop within the chitin-translocating channel, which acts as a 'gate lock' that prevents the substrate from leaving while directing the product polymer into the translocating channel for discharge to the extracellular side of the cell membrane. This work reveals the directional multistep mechanism of chitin biosynthesis and provides a structural basis for inhibition of chitin synthesis.

Biosynthesis of uridine diphosphate N-Acetylglucosamine: An underexploited pathway in the search for novel antibiotics?

Although the prevalence of antibiotic resistance is increasing at an alarming rate, there are a dwindling number of effective antibiotics available. Thus, the development of novel antibacterial agents should be of utmost importance. Peptidoglycan biosynthesis has been and is still an attractive source for antibiotic targets; however, there are several components that remain underexploited. In this review, we examine the enzymes involved in the biosynthesis of one such component, UDP-N-acetylglucosamine, an essential building block and precursor of bacterial peptidoglycan. Furthermore, given the presence of a similar biosynthesis pathway in eukaryotes, we discuss the current knowledge on the differences and similarities between the bacterial and eukaryotic enzymes. Finally, this review also summarises the recent advances made in the development of inhibitors targeting the bacterial enzymes.

Mechanisms of coordinating hyaluronan and glycosaminoglycan production by nucleotide sugars.

Hyaluronan is a versatile macromolecule capable of an exceptional range of functions from cushioning and hydration to dynamic signaling in development and disease. Because of its critical roles, hyaluronan production is regulated at multiple levels including epigenetic, transcriptional, and posttranslational control of the three hyaluronan synthase (HAS) enzymes. Precursor availability can dictate the rate and amount of hyaluronan synthesized and shed by the cells producing it. However, the nucleotide-activated sugar substrates for hyaluronan synthesis by HAS also participate in exquisitely fine-tuned cross-talking pathways that intersect with glycosaminoglycan production and central carbohydrate metabolism. Multiple UDP-sugars have alternative metabolic fates and exhibit coordinated and reciprocal allosteric control of enzymes within their biosynthetic pathways to preserve appropriate precursor ratios for accurate partitioning among downstream products, while also sensing and maintaining energy homeostasis. Since the dysregulation of nucleotide sugar and hyaluronan synthesis is associated with multiple pathologies, these pathways offer opportunities for therapeutic intervention. Recent structures of several key rate-limiting enzymes in the UDP-sugar synthesis pathways have offered new insights to the overall regulation of hyaluronan production by precursor fate decisions. The details of UDP-sugar control and the structural basis for underlying mechanisms are discussed in this review.

Silencing uridine diphosphate N-acetylglucosamine pyrophosphorylase gene impairs larval development in Henosepilachna vigintioctopunctata.

BACKGROUND: Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) diphosphorylase (UAP) catalyzes the formation of UDP-GlcNAc, the precursor for the production of chitin in ectodermally derived epidermal cells and midgut, for GlcNAcylation of proteins and for generation of glycosyl-phosphatidyl-inositol anchors in all tissues in Drosophila melanogaster. RESULTS: Here, we identified a putative HvUAP gene in Henosepilachna vigintioctopunctata. Knockdown of HvUAP at the second-, third- and fourth-instar stages impaired larval development. Most resultant HvUAP hypomorphs showed arrested development at the third-, fourth-instar larval or prepupal stages, and became paralyzed, depending on the age when treated. Some HvUAP-silenced larvae had weak and soft scoli. A portion of HvUAP-depleted beetles formed misshapen pupae. No HvUAP RNA interference pupae successfully emerged as adults. Dissection and microscopic observation revealed that knockdown of HvUAP affected gut growth and food ingestion, reduced cuticle thickness, and negatively affected the formation of newly generated cuticle layers during ecdysis. Furthermore, HvUAP deficiency inhibited development of the tracheal respiratory system and thinned tracheal taenidia. CONCLUSION: The phenotypical defects in HvUAP hypomorphs suggest that HvUAP is involved in the production of chitin. Moreover, our findings will enable the development of a double-stranded RNA-based pesticide to control H. vigintioctopunctata. © 2021 Society of Chemical Industry.

Activity of Lymphostatin, A Lymphocyte Inhibitory Virulence Factor of Pathogenic Escherichia coli, is Dependent on a Cysteine Protease Motif.

Lymphostatin (LifA) is a 366 kDa protein expressed by attaching & effacing Escherichia coli. It plays an important role in intestinal colonisation and inhibits the mitogen- and antigen-stimulated proliferation of lymphocytes and the synthesis of proinflammatory cytokines. LifA exhibits N-terminal homology with the glycosyltransferase domain of large clostridial toxins (LCTs). A DTD motif within this region is required for lymphostatin activity and binding of the sugar donor uridine diphosphate N-acetylglucosamine. As with LCTs, LifA also contains a cysteine protease motif (C1480, H1581, D1596) that is widely conserved within the YopT-like superfamily of cysteine proteases. By analogy with LCTs, we hypothesised that the CHD motif may be required for intracellular processing of the protein to release the catalytic N-terminal domain after uptake and low pH-stimulated membrane insertion of LifA within endosomes. Here, we created and validated a C1480A substitution mutant in LifA from enteropathogenic E. coli strain E2348/69. The purified protein was structurally near-identical to the wild-type protein. In bovine T lymphocytes treated with wild-type LifA, a putative cleavage product of approximately 140 kDa was detected. Appearance of the putative cleavage product was inhibited in a concentration-dependent manner by bafilomycin A1 and chloroquine, which inhibit endosome acidification. The cleavage product was not observed in cells treated with the C1480A mutant of LifA. Lymphocyte inhibitory activity of the purified C1480A protein was significantly impaired. The data indicate that an intact cysteine protease motif is required for cleavage of lymphostatin and its activity against T cells.

How the molecular weight affects the in vivo fate of exogenous hyaluronan delivered intravenously: A stable-isotope labelling strategy.

There is inconsistent information regarding the size effects of exogenously given hyaluronan on its in vivo fate. The data are often biased by the poor quality of hyaluronan and non-ideal labelling strategies used for resolving exogenous/endogenous hyaluronan, which only monitor the label and not hyaluronan itself. To overcome these drawbacks and establish the pharmacokinetics of intravenous hyaluronan in relation to its Mw, 13C-labelled HA of five Mws from 13.6-1562 kDa was prepared and administered to mice at doses 25-50 mg kg-1. The elimination efficiency increased with decreasing Mw. Low Mw hyaluronan was rapidly eliminated as small hyaluronan fragments in urine, while high Mw hyaluronan exhibited saturable kinetics and complete metabolization within 48 h. All tested Mws exhibited a similar uptake by liver cells and metabolization into activated sugars. 13C-labelling combined with LC-MS provides an excellent approach to elucidating in vivo fate and biological activities of hyaluronan.

Protein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1.

The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and via phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interferes with UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the interactions of the two GFAT-1 domains to control catalytic activity. Notably, Ser205 phosphorylation has two discernible effects: it lowers baseline GFAT-1 activity and abolishes UDP-GlcNAc feedback inhibition. PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1.

The folate cycle enzyme MTHFD2 induces cancer immune evasion through PD-L1 up-regulation.

Metabolic enzymes and metabolites display non-metabolic functions in immune cell signalling that modulate immune attack ability. However, whether and how a tumour's metabolic remodelling contributes to its immune resistance remain to be clarified. Here we perform a functional screen of metabolic genes that rescue tumour cells from effector T cell cytotoxicity, and identify the embryo- and tumour-specific folate cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2). Mechanistically, MTHFD2 promotes basal and IFN-γ-stimulated PD-L1 expression, which is necessary for tumourigenesis in vivo. Moreover, IFN-γ stimulates MTHFD2 through the AKT-mTORC1 pathway. Meanwhile, MTHFD2 drives the folate cycle to sustain sufficient uridine-related metabolites including UDP-GlcNAc, which promotes the global O-GlcNAcylation of proteins including cMYC, resulting in increased cMYC stability and PD-L1 transcription. Consistently, the O-GlcNAcylation level positively correlates with MTHFD2 and PD-L1 in pancreatic cancer patients. These findings uncover a non-metabolic role for MTHFD2 in cell signalling and cancer biology.

Characterization of two enzymes from Psychrobacter cryohalolentis that are required for the biosynthesis of an unusual diacetamido-d-sugar.

Psychrobacter cryohalolentis strain K5T is a Gram-negative organism first isolated in 2006. It has a complex O-antigen that contains, in addition to l-rhamnose and d-galactose, two diacetamido- and a triacetamido-sugar. The biochemical pathways for the production of these unusual sugars are presently unknown. Utilizing the published genome sequence of the organism, we hypothesized that the genes 0612, 0638, and 0637 encode for a 4,6-dehydratase, an aminotransferase, and an N-acetyltransferase, respectively, which would be required for the biosynthesis of one of the diacetamido-sugars, 2,4-diacetamido-2,4,6-trideoxy-d-glucose, starting from UDP-N-acetylglucosamine. Here we present functional and structural data on the proteins encoded by the 0638 and 0637 genes. The kinetic properties of these enzymes were investigated by a discontinuous HPLC assay. An X-ray crystallographic structure of 0638, determined in its external aldimine form to 1.3 Å resolution, demonstrated the manner in which the UDP ligand is positioned into the active site. It is strikingly different from that previously observed for PglE from Campylobacter jejuni, which functions on the same substrate. Four X-ray crystallographic structures were also determined for 0637 in various complexed states at resolutions between 1.3 and 1.55 Å. Remarkably, a tetrahedral intermediate mimicking the presumed transition state was trapped in one of the complexes. The data presented herein confirm the hypothesized functions of these enzymes and provide new insight into an unusual sugar biosynthetic pathway in Gram-negative bacteria. We also describe an efficient method for acetyl-CoA synthesis that allowed us to overcome its prohibitive cost for this analysis.

Metabolomics analysis reveals metabolic changes associated with trans-resveratrol treatment in experimental cryptorchidism mice.

This study aimed to analyse global metabolomic changes associated with trans-resveratrol (RSV) treatment in mice with cryptorchidism using untargeted metabolomics. Cryptorchidism was established surgically in Kunming mice, which were then treated with 20µg g-1 day-1, s.c., RSV for 35 consecutive days. Typical manifestations of spermatogenesis arrest were seen in mice with cryptorchidism, and RSV treatment for 35 days restored spermatogenesis. Liquid chromatography-tandem mass spectrometry was used to profile the metabolome of testes from mice in the control (non-cryptorchid, untreated), cryptorchid and RSV-treated cryptorchid groups. In all, 1386 and 179 differential metabolites were detected in the positive and negative modes respectively. Seven and six potential biomarkers were screened for spermatogenesis arrest and restoration respectively. Pathway analysis showed changes in 197 metabolic pathways. The hexosamine biosynthesis pathway was inhibited in the cryptorchid group, which probably resulted in a decrease in the end product, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Immunoblot analysis showed that total testicular protein O-linked ß-N-acetylglucosamine glycosylation was related to spermatogenesis arrest, further indicating a decrease in UDP-GlcNAc in the cryptorchid group. Thus, untargeted metabolomics revealed the biochemical pathways associated with the restoration of metabolic status in the cryptorchid group following RSV treatment and the findings could be used to monitor the response to RSV treatment. This study provides a meaningful foundation for the future clinical application of RSV in the treatment of spermatogenesis dysfunction.

The hexosamine biosynthetic pathway and cancer: Current knowledge and future therapeutic strategies.

The hexosamine biosynthetic pathway (HBP) is a glucose metabolism pathway that results in the synthesis of a nucleotide sugar UDP-GlcNAc, which is subsequently used for the post-translational modification (O-GlcNAcylation) of intracellular proteins that regulate nutrient sensing and stress response. The HBP is carried out by a series of enzymes, many of which have been extensively implicated in cancer pathophysiology. Increasing evidence suggests that elevated activation of the HBP may act as a cancer biomarker. Inhibition of HBP enzymes could suppress tumor cell growth, modulate the immune response, reduce resistance, and sensitize tumor cells to conventional cancer therapy. Therefore, targeting the HBP may serve as a novel strategy for treating cancer patients. Here, we review the current findings on the significance of HBP enzymes in various cancers and discuss future approaches for exploiting HBP inhibition for cancer treatment.

Cell Energy Metabolism and Hyaluronan Synthesis.

Hyaluronan (HA) is a linear glycosaminoglycan (GAG) of extracellular matrix (ECM) synthesized by three hyaluronan synthases (HASes) at the plasma membrane using uridine diphosphate (UDP)-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc) as substrates. The production of HA is mainly regulated by hyaluronan synthase 2 (HAS2), that can be controlled at different levels, from epigenetics to transcriptional and post-translational modifications. HA biosynthesis is an energy-consuming process and, along with HA catabolism, is strongly connected to the maintenance of metabolic homeostasis. The cytoplasmic pool of UDP-sugars is critical for HA synthesis. UDP-GlcNAc is an important nutrient sensor and serves as donor substrate for the O-GlcNAcylation of many cytosolic proteins, including HAS2. This post-translational modification stabilizes HAS2 in the membrane and increases HA production. Conversely, HAS2 can be phosphorylated by AMP activated protein kinase (AMPK), a master metabolic regulator activated by low ATP/AMP ratios, which inhibits HA secretion. Similarly, HAS2 expression and the deposition of HA within the pericellular coat are inhibited by sirtuin 1 (SIRT1), another important energetic sensor, confirming the tight connection between nutrients availability and HA metabolism.

Accelerated human epidermal turnover driven by increased hyaluronan production.

BACKGROUND: Hyaluronan (HA) is an essential component of extracellular matrix in the skin, but its functions in the epidermis remain elusive. OBJECTIVE: We examined the interaction of increased HA production mediated by 1-ethyl-ß-N-acetylglucosaminide (ß-NAG2), a newly developed highly selective inducer of HA production which is intracellularly converted to UDP-N-acetylglucosamine, a substrate of HA, with epidermal proliferation and differentiation. METHODS: The amount, molecular size and epidermal tissue distribution of HA and expression of CD44, a cell surface receptor for HA, were analyzed in ß-NAG2-treated organ cultured human skin, reconstructed human skin equivalents or cultured human skin keratinocytes. The relationship between HA and epidermal proliferation or differentiation was examined. RESULTS: ß-NAG2 significantly increased HA production in the epidermis of skin explants or skin equivalents without affecting molecular size of HA (>2000 kDa) or CD44 mRNA expression. Histochemical experiments revealed that ß-NAG2 enhances HA signals in the basal to granular layers of the epidermis of skin equivalents, accompanying increased epidermal stratification. Immunohistochemical experiments demonstrated that signals of Ki67, transglutaminase 1 and filaggrin are increased in ß-NAG2-treated skin equivalents, and these observations were confirmed by the data showing that mRNA expression of PCNA, transglutaminase 1 (TGM1) and filaggrin (FLG) is significantly up-regulated by ß-NAG2 in skin equivalents. Importantly, blockade of HA production by inhibiting conversion of ß-NAG2 to UDP-NAG abolished ß-NAG2-mediated up-regulation of PCNA, TGM1 and FLG mRNA expression in cultured keratinocytes. CONCLUSION: These results suggest that increased epidermal HA production plays a key role in epidermal morphogenesis and homeostasis by accelerating keratinocyte proliferation and differentiation.

Stem cell fate determination through protein O-GlcNAcylation.

Embryonic and adult stem cells possess the capability of self-renewal and lineage-specific differentiation. The intricate balance between self-renewal and differentiation is governed by developmental signals and cell-type-specific gene regulatory mechanisms. A perturbed intra/extracellular environment during lineage specification could affect stem cell fate decisions resulting in pathology. Growing evidence demonstrates that metabolic pathways govern epigenetic regulation of gene expression during stem cell fate commitment through the utilization of metabolic intermediates or end products of metabolic pathways as substrates for enzymatic histone/DNA modifications. UDP-GlcNAc is one such metabolite that acts as a substrate for enzymatic mono-glycosylation of various nuclear, cytosolic, and mitochondrial proteins on serine/threonine amino acid residues, a process termed protein O-GlcNAcylation. The levels of GlcNAc inside the cells depend on the nutrient availability, especially glucose. Thus, this metabolic sensor could modulate gene expression through O-GlcNAc modification of histones or other proteins in response to metabolic fluctuations. Herein, we review evidence demonstrating how stem cells couple metabolic inputs to gene regulatory pathways through O-GlcNAc-mediated epigenetic/transcriptional regulatory mechanisms to govern self-renewal and lineage-specific differentiation programs. This review will serve as a primer for researchers seeking to better understand how O-GlcNAc influences stemness and may catalyze the discovery of new stem-cell-based therapeutic approaches.

Antitoxin ε Reverses Toxin ζ-Facilitated Ampicillin Dormants.

Toxin-antitoxin (TA) modules are ubiquitous in bacteria, but their biological importance in stress adaptation remains a matter of debate. The inactive ζ-ε2-ζ TA complex is composed of one labile ε2 antitoxin dimer flanked by two stable ζ toxin monomers. Free toxin ζ reduces the ATP and GTP levels, increases the (p)ppGpp and c-di-AMP pool, inactivates a fraction of uridine diphosphate-N-acetylglucosamine, and induces reversible dormancy. A small subpopulation, however, survives toxin action. Here, employing a genetic orthogonal control of ζ and ε levels, the fate of bacteriophage SPP1 infection was analyzed. Toxin ζ induces an active slow-growth state that halts SPP1 amplification, but it re-starts after antitoxin expression rather than promoting abortive infection. Toxin ζ-induced and toxin-facilitated ampicillin (Amp) dormants have been revisited. Transient toxin ζ expression causes a metabolic heterogeneity that induces toxin and Amp dormancy over a long window of time rather than cell persistence. Antitoxin ε expression, by reversing ζ activities, facilitates the exit of Amp-induced dormancy both in rec+ and recA cells. Our findings argue that an unexploited target to fight against antibiotic persistence is to disrupt toxin-antitoxin interactions.

Structural characterization of a nonhydrolyzing UDP-GlcNAc 2-epimerase from Neisseria meningitidis serogroup A.

Bacterial nonhydrolyzing UDP-N-acetylglucosamine 2-epimerases catalyze the reversible interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). UDP-ManNAc is an important intermediate in the biosynthesis of certain cell-surface polysaccharides, including those in some pathogenic bacteria, such as Neisseria meningitidis and Streptococcus pneumoniae. Many of these epimerases are allosterically regulated by UDP-GlcNAc, which binds adjacent to the active site and is required to initiate UDP-ManNAc epimerization. Here, two crystal structures of UDP-N-acetylglucosamine 2-epimerase from Neisseria meningitidis serogroup A (NmSacA) are presented. One crystal structure is of the substrate-free enzyme, while the other structure contains UDP-GlcNAc substrate bound to the active site. Both structures form dimers as seen in similar epimerases, and substrate binding to the active site induces a large conformational change in which two Rossmann-like domains clamp down on the substrate. Unlike other epimerases, NmSacA does not require UDP-GlcNAc to instigate the epimerization of UDP-ManNAc, although UDP-GlcNAc was found to enhance the rate of epimerization. In spite of the conservation of residues involved in binding the allosteric UDP-GlcNAc observed in similar UDP-GlcNAc 2-epimerases, the structures presented here do not contain UDP-GlcNAc bound in the allosteric site. These structural results provide additional insight into the mechanism and regulation of this critical enzyme and improve the structural understanding of the ability of NmSacA to epimerize modified substrates.

Uridine diphosphate N-acetylglucosamine orchestrates the interaction of GlmR with either YvcJ or GlmS in Bacillus subtilis.

In bacteria, glucosamine-6-phosphate (GlcN6P) synthase, GlmS, is an enzyme required for the synthesis of Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a precursor of peptidoglycan. In Bacillus subtilis, an UDP-GlcNAc binding protein, GlmR (formerly YvcK), essential for growth on non-glycolytic carbon sources, has been proposed to stimulate GlmS activity; this activation could be antagonized by UDP-GlcNAc. Using purified proteins, we demonstrate that GlmR directly stimulates GlmS activity and the presence of UDP-GlcNAc (at concentrations above 0.1 mM) prevents this regulation. We also showed that YvcJ, whose gene is associated with yvcK (glmR), interacts with GlmR in an UDP-GlcNAc dependent manner. Strains producing GlmR variants unable to interact with YvcJ show decreased transformation efficiency similar to that of a yvcJ null mutant. We therefore propose that, depending on the intracellular concentration of UDP-GlcNAc, GlmR interacts with either YvcJ or GlmS. When UDP-GlcNAc concentration is high, this UDP-sugar binds to YvcJ and to GlmR, blocking the stimulation of GlmS activity and driving the interaction between GlmR and YvcJ to probably regulate the cellular role of the latter. When the UDP-GlcNAc level is low, GlmR does not interact with YvcJ and thus does not regulate its cellular role but interacts with GlmS to stimulate its activity.

PelX is a UDP-N-acetylglucosamine C4-epimerase involved in Pel polysaccharide-dependent biofilm formation.

Pel is a GalNAc-rich bacterial polysaccharide that contributes to the structure and function of Pseudomonas aeruginosa biofilms. The pelABCDEFG operon is highly conserved among diverse bacterial species, and Pel may therefore be a widespread biofilm determinant. Previous annotation of pel gene clusters has helped us identify an additional gene, pelX, that is present adjacent to pelABCDEFG in >100 different bacterial species. The pelX gene is predicted to encode a member of the short-chain dehydrogenase/reductase (SDR) superfamily, but its potential role in Pel-dependent biofilm formation is unknown. Herein, we have used Pseudomonas protegens Pf-5 as a model to elucidate PelX function as Pseudomonas aeruginosa lacks a pelX homologue in its pel gene cluster. We found that P. protegens forms Pel-dependent biofilms; however, despite expression of pelX under these conditions, biofilm formation was unaffected in a ΔpelX strain. This observation led us to identify a pelX paralogue, PFL_5533, which we designate here PgnE, that appears to be functionally redundant to pelX In line with this, a ΔpelX ΔpgnE double mutant was substantially impaired in its ability to form Pel-dependent biofilms. To understand the molecular basis for this observation, we determined the structure of PelX to 2.1 Å resolution. The structure revealed that PelX resembles UDP-GlcNAc C4-epimerases. Using 1H NMR analysis, we show that PelX catalyzes the epimerization between UDP-GlcNAc and UDP-GalNAc. Our results indicate that Pel-dependent biofilm formation requires a UDP-GlcNAc C4-epimerase that generates the UDP-GalNAc precursors required by the Pel synthase machinery for polymer production.