The host mannose-6-phosphate pathway and viral infection.

Viruses, despite their simple structural composition, engage in intricate and complex interactions with their hosts due to their parasitic nature. A notable demonstration of viral behavior lies in their exploitation of lysosomes, specialized organelles responsible for the breakdown of biomolecules and clearance of foreign substances, to bolster their own replication. The man-nose-6-phosphate (M6P) pathway, crucial for facilitating the proper transport of hydrolases into lysosomes and promoting lysosome maturation, is frequently exploited for viral manipulation in support of replication. Recently, the discovery of lysosomal enzyme trafficking factor (LYSET) as a pivotal regulator within the lysosomal M6P pathway has introduced a fresh perspective on the intricate interplay between viral entry and host factors. This groundbreaking revelation illuminates unexplored dimensions of these interactions. In this review, we endeavor to provide a thorough overview of the M6P pathway and its intricate interplay with viral factors during infection. By consolidating the current understanding in this field, our objective is to establish a valuable reference for the development of antiviral drugs that selectively target the M6P pathway.

Comparative site-specific N-glycoproteome analysis reveals aberrant N-glycosylation and gives insights into mannose-6-phosphate pathway in cancer.

N-glycosylation is implicated in cancers and aberrant N-glycosylation is recognized as a hallmark of cancer. Here, we mapped and compared the site-specific N-glycoproteomes of colon cancer HCT116 cells and isogenic non-tumorigenic DNMT1/3b double knockout (DKO1) cells using Fbs1-GYR N-glycopeptide enrichment technology and trapped ion mobility spectrometry. Many significant changes in site-specific N-glycosylation were revealed, providing a molecular basis for further elucidation of the role of N-glycosylation in protein function. HCT116 cells display hypersialylation especially in cell surface membrane proteins. Both HCT116 and DKO1 show an abundance of paucimannose and 80% of paucimannose-rich proteins are annotated to reside in exosomes. The most striking N-glycosylation alteration was the degree of mannose-6-phosphate (M6P) modification. N-glycoproteomic analyses revealed that HCT116 displays hyper-M6P modification, which was orthogonally validated by M6P immunodetection. Significant observed differences in N-glycosylation patterns of the major M6P receptor, CI-MPR in HCT116 and DKO1 may contribute to the hyper-M6P phenotype of HCT116 cells. This comparative site-specific N-glycoproteome analysis provides a pool of potential N-glycosylation-related cancer biomarkers, but also gives insights into the M6P pathway in cancer.

Structural investigation of a thermostable 1,2-ß-mannobiose phosphorylase from Thermoanaerobacter sp. X-514.

1,2-ß-Mannobiose phosphorylases (1,2-ß-MBPs) from glycoside hydrolase 130 (GH130) family are important bio-catalysts in glycochemistry applications owing to their ability in synthesizing oligomannans. Here, we report the crystal structure of a thermostable 1,2-ß-MBP from Thermoanaerobacter sp. X-514 termed Teth514_1789 to reveal the molecular basis of its higher thermostability and mechanism of action. We also solved the enzyme complexes of mannose, mannose-1-phosphate (M1P) and 1,4-ß-mannobiose to manifest the enzyme-substrate interaction networks of three main subsites. Notably, a Zn ion that should be derived from crystallization buffer was found in the active site and coordinates the phosphate moiety of M1P. Nonetheless, this Zn-coordination should reflect an inhibitory status as supplementing Zn severely impairs the enzyme activity. These results indicate that the effects of metal ions should be taken into consideration when applying Teth514_1789 and other related enzymes. Based on the structure, a reliable model of Teth514_1788 that shares 61.7% sequence identity to Teth514_1789 but displays a different substrate preference was built. Analyzing the structural features of these two closely related enzymes, we hypothesized that the length of a loop fragment that covers the entrance of the catalytic center might regulate the substrate selectivity. In conclusion, these information provide in-depth understanding of GH130 1,2-ß-MBPs and should serve as an important guidance for enzyme engineering for further applications.

Simultaneous enrichment and separation of neutral and sialyl glycopeptides of SARS-CoV-2 spike protein enabled by dual-functionalized Ti-IMAC material.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a serious threat to human health all over the world. The development of effective vaccines has been focusing on the spike (S) glycoprotein, which mediates viral invasion to human cells through its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor. In this work, we perform analytical characterization of N- and O-linked glycosylation of the SARS-CoV-2 S glycoprotein. We explore the novel use of dual-functionalized titanium (IV)-immobilized metal affinity chromatography (Ti-IMAC) material for simultaneous enrichment and separation of neutral and sialyl glycopeptides of a recombinant SARS-CoV-2 S glycoprotein from HEK293 cells. This strategy helps eliminate signal suppression from neutral glycopeptides for the detection of sialyl glycopeptides and improves the glycoform coverage of the S protein. We profiled 19 of its 22 potential N-glycosylated sites with 398 unique glycoforms using the dual-functional Ti-IMAC approach, which exhibited improvement of coverage by 1.6-fold compared to the conventional hydrophilic interaction chromatography (HILIC) glycopeptide enrichment method. We also identified O-linked glycosylation site that was not found using the conventional HILIC approach. In addition, we reported on the identification of mannose-6-phosphate (M6P) glycosylation, which substantially expands the current knowledge of the spike protein's glycosylation landscape and enables future investigation into the influence of M6P glycosylation of the spike protein on its cell entry.

Multivalent, Stabilized Mannose-6-Phosphates for the Targeted Delivery of Toll-Like Receptor Ligands and Peptide Antigens.

Mannose-6-phosphate (M6P) is recognized by the mannose-6-phosphate receptor and plays an important role in the transport of cargo to the endosomes, making it an attractive tool to improve endosomal trafficking of vaccines. We describe herein the assembly of peptide antigen conjugates carrying clusters of mannose-6-C-phosphonates (M6Po). The M6Po's are stable M6P mimics that are resistant to cleavage of the phosphate group by endogenous phosphatases. Two different strategies for the incorporation of the M6Po clusters in the conjugate have been developed: the first relies on a "post-assembly" click approach employing an M6Po bearing an alkyne functionality; the second hinges on an M6Po C-glycoside amino acid building block that can be used in solid-phase peptide synthesis. The generated conjugates were further equipped with a TLR7 ligand to stimulate dendritic cell (DC) maturation. While antigen presentation is hindered by the presence of the M6Po clusters, the incorporation of the M6Po clusters leads to increased activation of DCs, thus demonstrating their potential in improving vaccine adjuvanticity by intraendosomally active TLR ligands.

Dual enzyme responsive mannose-6-phosphate based vesicle for controlled lysosomal delivery.

Dual enzyme responsive stable biomimetic vesicles composed of mannose-6-phosphate lipid can encapsulate and deliver dual dye/drug and protein/enzyme exclusively to the lysosome in HEK-293 cells. The release of the cargo from the vesicles can be temporally controlled due to the enzyme responsive morphology change of the M6P lipid assembly.

Structure of the Human Cation-Independent Mannose 6-Phosphate/IGF2 Receptor Domains 7-11 Uncovers the Mannose 6-Phosphate Binding Site of Domain 9.

The cation-independent mannose 6-phosphate (M6P)/Insulin-like growth factor-2 receptor (CI-MPR/IGF2R) is an ∼300 kDa transmembrane protein responsible for trafficking M6P-tagged lysosomal hydrolases and internalizing IGF2. The extracellular region of the CI-MPR has 15 homologous domains, including M6P-binding domains (D) 3, 5, 9, and 15 and IGF2-binding domain 11. We have focused on solving the first structures of human D7-10 within two multi-domain constructs, D9-10 and D7-11, and provide the first high-resolution description of the high-affinity M6P-binding D9. Moreover, D9 stabilizes a well-defined hub formed by D7-11 whereby two penta-domains intertwine to form a dimeric helical-type coil via an N-glycan bridge on D9. Remarkably the D7-11 structure matches an IGF2-bound state of the receptor, suggesting this may be an intrinsically stable conformation at neutral pH. Interdomain clusters of histidine and proline residues may impart receptor rigidity and play a role in structural transitions at low pH.

Crystal Structure of the Mannose-6-Phosphate Uncovering Enzyme.

Most lysosomal hydrolytic enzymes reach their destination via the mannose-6-phosphate (M6P) pathway. The enzyme N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (NAGPA, or "uncovering enzyme") catalyzes the second step in the M6P tag formation, namely the removal of the masking N-acetylglucosamine (GlcNAc) portion. Defects in this protein are associated with non-syndromic stuttering. To gain a better understanding of the function and regulation of this enzyme, we determined its crystal structure. The propeptide binds in a groove on the globular catalytic domain, blocking active site access. High-affinity substrate binding is enabled by a conformational switch in an active site loop. The protein recognizes the GlcNAc and phosphate portions of its substrate, but not the mannose moiety of the glycan. Based on enzymatic and 1H-NMR analysis, a catalytic mechanism is proposed. Crystallographic and solution scattering analyses suggest that the C-terminal domain forms a long flexible stem that extends the enzyme away from the Golgi membrane.

Expression, purification, and characterization of human mannose-6-phosphate receptor - Extra cellular domain from a stable cell line utilizing a small molecule biomimetic of the mannose-6-phosphate moiety.

The cation-independent mannose-6-phosphate receptor (CI-M6PR, aka insulin-like growth factor II receptor or IGFIIR) is a membrane protein that plays a central role in the trafficking of lysosomal acid hydrolases into lysosomes via mannose-6-phosphate (M6P) binding domains. In order to maintain cellular metabolic/catabolic homeostasis, newly synthesized lysosomal acid hydrolases are required to bind to M6PR for transit. Acid hydrolases secreted by cells can also be internalized via M6PR residing on the cell membrane and are transported to the lysosomes, a feature that enables enzyme replacement therapy for the treatment of several lysosomal storage disorders. Therefore, a thorough characterization of this receptor is critical to the development of lysosomal enzyme-based therapeutics that utilize M6PR for drug delivery to the lysosome. However, the extracellular domain (ECD) of M6PR is highly complex, containing 15-mannose receptor homology (MRH) domains. In addition, homodimerization of the receptor can occur at the membrane, making its characterization challenging. In this study, a novel human M6PR (hM6PR)-overexpressing cell line originally established for hM6PR cellular uptake assay was utilized for production of hM6PR-ECD, and a novel small molecule biomimetic (aminophenyl-M6P) affinity resin was developed for the purification of M6PR-ECD. The affinity-purified hM6PR-ECD was monomeric, contained 14 intact MRH domains (1-14) and a partial MRH domain 15, and was successfully employed in ELISA-based and surface plasmon resonance-based binding assays to demonstrate its ligand-binding functionality, making it suitable for the evaluation of biotherapeutics that utilize M6PR for cellular internalization.

Dual-Functional Titanium(IV) Immobilized Metal Affinity Chromatography Approach for Enabling Large-Scale Profiling of Protein Mannose-6-Phosphate Glycosylation and Revealing Its Predominant Substrates.

Mannose-6-phosphate (M6P) glycosylation is an important post-translational modification (PTM) and plays a crucial role in transferring lysosomal hydrolases to lysosome, and is involved in several other biological processes. Aberrant M6P modifications have been implicated in lysosomal storage diseases and numerous other disorders including Alzheimer's disease and cancer. Research on profiling of intact M6P glycopeptides remains challenging due to its extremely low stoichiometry. Here we propose a dual-mode affinity approach to enrich M6P glycopeptides by dual-functional titanium(IV) immobilized metal affinity chromatography [Ti(IV)-IMAC] materials. In combination with state-of-the-art mass spectrometry and database search engine, we profiled 237 intact M6P glycopeptides corresponding to 81 M6P glycoproteins in five types of tissues in mouse, representing the first large-scale profiling of M6P glycosylation in mouse samples. The analysis of M6P glycoforms revealed the predominant glycan substrates of this PTM. Gene ontology analysis showed that overrepresented M6P glycoproteins were lysosomal-associated proteins. However, there were still substantial M6P glycoproteins that possessed different subcellular locations and molecular functions. Deep mining of their roles implicated in lysosomal and nonlysosomal function can provide new insights into functional roles of this important yet poorly studied modification.

Efficient Photodynamic Therapy of Prostate Cancer Cells through an Improved Targeting of the Cation-Independent Mannose 6-Phosphate Receptor.

The aim of the present work is the development of highly efficient targeting molecules to specifically address mesoporous silica nanoparticles (MSNs) designed for the photodynamic therapy (PDT) of prostate cancer. We chose the strategy to develop a novel compound that allows the improvement of the targeting of the cation-independent mannose 6-phosphate receptor, which is overexpressed in prostate cancer. This original sugar, a dimannoside-carboxylate (M6C-Man) grafted on the surface of MSN for PDT applications, leads to a higher endocytosis and thus increases the efficacy of MSNs.

Unravelling the Specificity of Laminaribiose Phosphorylase from Paenibacillus sp. YM-1 towards Donor Substrates Glucose/Mannose 1-Phosphate by Using X-ray Crystallography and Saturation Transfer Difference NMR Spectroscopy.

Glycoside phosphorylases (GPs) carry out a reversible phosphorolysis of carbohydrates into oligosaccharide acceptors and the corresponding sugar 1-phosphates. The reversibility of the reaction enables the use of GPs as biocatalysts for carbohydrate synthesis. Glycosyl hydrolase family 94 (GH94), which only comprises GPs, is one of the most studied GP families that have been used as biocatalysts for carbohydrate synthesis, in academic research and in industrial production. Understanding the mechanism of GH94 enzymes is a crucial step towards enzyme engineering to improve and expand the applications of these enzymes in synthesis. In this work with a GH94 laminaribiose phosphorylase from Paenibacillus sp. YM-1 (PsLBP), we have demonstrated an enzymatic synthesis of disaccharide 1 (ß-d-mannopyranosyl-(1→3)-d-glucopyranose) by using a natural acceptor glucose and noncognate donor substrate α-mannose 1-phosphate (Man1P). To investigate how the enzyme recognises different sugar 1-phosphates, the X-ray crystal structures of PsLBP in complex with Glc1P and Man1P have been solved, providing the first molecular detail of the recognition of a noncognate donor substrate by GPs, which revealed the importance of hydrogen bonding between the active site residues and hydroxy groups at C2, C4, and C6 of sugar 1-phosphates. Furthermore, we used saturation transfer difference NMR spectroscopy to support crystallographic studies on the sugar 1-phosphates, as well as to provide further insights into the PsLBP recognition of the acceptors and disaccharide products.

Synthesis of high-mannose oligosaccharides containing mannose-6-phosphate residues using regioselective glycosylation.

Molecular recognition of mannose-6-phosphate (M6P)-modified oligosaccharides by transmembrane M6P receptors is a key signaling event in lysosomal protein trafficking in vivo. Access to M6P-containing high-mannose N-glycans is essential to achieving a thorough understanding of the M6P ligand-receptor recognition process. Herein we report the application of a versatile and reliable chemical strategy to prepare asymmetric di-antennary M6P-tagged high-mannose oligosaccharides in >20% overall yield and in high purity (>98%). Regioselective chemical glycosylation coupled with effective phosphorylation and product purification protocols were applied to rapidly assemble these oligosaccharides. The development of this synthetic strategy simplifies the preparation of M6P-tagged high-mannose oligosaccharides, which will improve access to these compounds to study their structures and biological functions.

A Glycoengineered Enzyme with Multiple Mannose-6-Phosphates Is Internalized into Diseased Cells to Restore Its Activity in Lysosomes.

In this study we developed an efficient method to prepare glycoengineered ß-N-acetylhexosaminidase containing multiple mannose-6-phosphates (M6Ps) by combining genetic code expansion with bioorthogonal ligation techniques. We found that multiple M6P-conjugated enzymes were produced with a high efficiency by using combined techniques. Importantly, glycoengineered enzymes entered lysosomes of patient-derived primary cells, which lack endogenous lysosomal ß-N-acetylhexosaminidase, more readily than commercialized human ß-hexosaminidase. Moreover, glycoengineered enzymes successfully removed GM2-ganglioside stored in lysosomes of diseased cells, indicating that its activity is restored in diseased cells. We also synthesized and applied a lysosome-targeting fluorogenic substrate to monitor endogenous and supplemental glycoengineered ß-N-acetylhexosaminidase activities in lysosomes. The results of this study indicate that the present strategy, which relies on genetic code expansion and bioorthogonal ligation techniques, is highly attractive to generate multi-M6P-containing lysosomal enzymes that can be used to study lysosomal storage disorders associated with lysosomal enzyme deficiencies.

Discovery of a Kojibiose Phosphorylase in Escherichia coli K-12.

The substrate profiles for three uncharacterized enzymes (YcjM, YcjT, and YcjU) that are expressed from a cluster of 12 genes ( ycjM-W and ompG) of unknown function in Escherichia coli K-12 were determined. Through a comprehensive bioinformatic and steady-state kinetic analysis, the catalytic function of YcjT was determined to be kojibiose phosphorylase. In the presence of saturating phosphate and kojibiose (α-(1,2)-d-glucose-d-glucose), this enzyme catalyzes the formation of d-glucose and ß-d-glucose-1-phosphate ( kcat = 1.1 s-1, Km = 1.05 mM, and kcat/ Km = 1.12 × 103 M-1 s-1). Additionally, it was also shown that in the presence of ß-d-glucose-1-phosphate, YcjT can catalyze the formation of other disaccharides using 1,5-anhydro-d-glucitol, l-sorbose, d-sorbitol, or l-iditol as a substitute for d-glucose. Kojibiose is a component of cell wall lipoteichoic acids in Gram-positive bacteria and is of interest as a potential low-calorie sweetener and prebiotic. YcjU was determined to be a ß-phosphoglucomutase that catalyzes the isomerization of ß-d-glucose-1-phosphate ( kcat = 21 s-1, Km = 18 µM, and kcat/ Km = 1.1 × 106 M-1 s-1) to d-glucose-6-phosphate. YcjU was also shown to exhibit catalytic activity with ß-d-allose-1-phosphate, ß-d-mannose-1-phosphate, and ß-d-galactose-1-phosphate. YcjM catalyzes the phosphorolysis of α-(1,2)-d-glucose-d-glycerate with a kcat = 2.1 s-1, Km = 69 µM, and kcat/ Km = 3.1 × 104 M-1 s-1.

Mannose 6-phosphate-dependent lysosomal enzyme targeting in hydra: a biochemical, immunological and structural elucidation.

Mannose 6-phosphate (M6P)-dependent lysosomal enzyme targeting to endosome/lysosome complex is poorly understood among lower invertebrates. So far, only a M6P-independent lysosomal enzyme sorting protein, named LERP, has been described in Drosophila. Here, we have identified mannose 6-phosphate receptor (MPR) homologues in Hydra vulgaris, a basal Cnidarian, at genome level and further purified a cation-dependent MPR-like protein from hydra using affinity chromatography. Structural comparisons of hydra MPRs with mammalian MPRs confirm that the residues important for interacting with the M6P ligand are conserved. Based on our results, we report for the first time the occurrence of MPR-related proteins and M6P-dependent lysosomal enzyme targeting in H. vulgaris.

Selective induction of apoptosis in MCF7 cancer-cell by targeted liposomes functionalised with mannose-6-phosphate.

Liposomes are versatile platforms to carry anticancer drugs in targeted drug delivery; they can be surface modified by different strategies and, when coupled with targeting ligands, are able to increase cellular internalisation and organelle-specific drug delivery. An interesting strategy of antitumoral therapy could involve the use of lysosomotropic ligand-targeted liposomes loaded with molecules, which can induce lysosomal membrane permeabilization (LMP), leakage of cathepsins into the cytoplasm and subsequent apoptosis. We have previously demonstrated the ability of liposomes functionalised with a mannose-6-phosphate to reach lysosomes; in this research we compare the behaviour of M6P-modified and non-functionalised liposomes in MCF7 tumour cell and in HDF normal cells. With this aim, we first demonstrated by Western blotting the overexpression of mannose-6-phosphate/insulin-like growth factor (M6P/IGF-II) receptor in MCF7. Then, we prepared calcein-loaded liposomes and we revealed the increased uptake of M6P-functionalised liposomes in MCF7 cells respect to HDF cells by flow cytometry analysis. Finally, we loaded functionalised and not functionalised liposomes with N-hexanoyl-d-erythro-sphingosine (C6Cer), able to initiate LMP-induced apoptosis; after having studied the stability of both vesicles in the presence of serum by Dynamic Light Scattering and Spectrophotometric turbidity measurements, we showed that ceramide-loaded M6P-liposomes significantly increased apoptosis in MCF7 with respect to HDF cells.

Four unreported types of glycans containing mannose-6-phosphate are heterogeneously attached at three sites (including newly found Asn 233) to recombinant human acid alpha-glucosidase that is the only approved treatment for Pompe disease.

Myozyme is a recombinant human acid alpha-glucosidase (rhGAA) that is currently the only drug approved for treating Pompe disease, and its low efficacy means that a high dose is required. Mannose-6-phosphate (M6P) glycosylation on rhGAA is a key factor influencing lysosomal enzyme targeting and the efficacy of enzyme replacement therapy (ERT); however, its complex structure and relatively small quantity still remain to be characterized. This study investigated M6P glycosylation on rhGAA using liquid chromatography (LC)-electrospray ionization (ESI)-high-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS). The glycans released from rhGAA were labeled with procainamide to improve mass ionization efficiency and the sensitivity of MS/MS. The relative quantities (%) of 78 glycans were obtained, and 1.0% of them were glycans containing M6P (M6P glycans). These were categorized according to their structure into 4 types: 3 newly found ones, comprising high-mannose-type M6P glycans capped with N-acetylglucosamine (GlcNAc) (2 variants, 17.5%), hybrid-type M6P glycans (2 variants, 11.2%), and hybrid-type M6P glycans capped with GlcNAc (3 variants, 6.9%), as well as high-mannose-type M6P glycans (3 variants, 64.4%). HCD-MS/MS spectra identified six distinctive M6P-derived oxonium ions. The glycopeptides obtained from protease-digested rhGAA were analyzed using nano-LC-ESI-HCD-MS/MS, and the extracted-ion chromatograms of M6P-derived oxonium ions confirmed three M6P glycosylation sites comprising Asn 140, Asn 233 (newly found), and Asn 470 attached heterogeneously to nine M6P glycans (two types), eight M6P glycans (four types), and seven M6P glycans (two types), respectively. This is the first study of rhGAA to differentiate M6P glycans and identify their attachment sites, despite rhGAA already being an approved drug for Pompe disease.

Efficient therapy for refractory Pompe disease by mannose 6-phosphate analogue grafting on acid α-glucosidase.

Pompe disease is a rare disorder due to deficiency of the acid α-glucosidase (GAA) treated by enzyme replacement therapy. The present authorized treatment with rhGAA, the recombinant human enzyme, provides an important benefit in the infantile onset; however, the juvenile and adult forms of the disease corresponding to >80% of the patients are less responsive to this treatment. This resistance has been mainly attributed to an insufficiency of mannose 6-phosphate residues in rhGAA to address lysosomes through the cation-independent mannose 6-phosphate receptor (CI-M6PR). As yet, several attempts to improve the enzyme delivery by increasing the number of mannose 6-phosphate on the enzyme were poorly effective on the late onset form of the disease. Here, we show that chemical conjugation of a synthetic analogue of the mannose 6-phosphate, named AMFA, onto rhGAA improves the affinity for CI-M6PR and the uptake of the enzyme in fibroblasts and myoblasts of adult Pompe patients. More importantly, only the conjugated rhGAA-AMFA was effective in aged Pompe mice when compared to rhGAA. Weekly treatment with 5-20mg·kg-1 rhGAA-AMFA provided major improvements of the motor function and of the myofiber structure, whereas rhGAA was inactive. Finally, AMFA addition did not induce supplementary immune response to the enzyme. This modified enzyme, displaying a muscle recovery in aged Pompe mice that was never attained before, could be considered as a potential therapy for the late onset Pompe disease.

Glucose-1-phosphate uridylyltransferase from Erwinia amylovora: Activity, structure and substrate specificity.

Erwinia amylovora, a Gram-negative plant pathogen, is the causal agent of Fire Blight, a contagious necrotic disease affecting plants belonging to the Rosaceae family, including apple and pear. E. amylovora is highly virulent and capable of rapid dissemination in orchards; effective control methods are still lacking. One of its most important pathogenicity factors is the exopolysaccharide amylovoran. Amylovoran is a branched polymer made by the repetition of units mainly composed of galactose, with some residues of glucose, glucuronic acid and pyruvate. E. amylovora glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, EC 2.7.7.9) has a key role in amylovoran biosynthesis. This enzyme catalyses the production of UDP-glucose from glucose-1-phosphate and UTP, which the epimerase GalE converts into UDP-galactose, the main building block of amylovoran. We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-d-glucosamine 1-phosphate, 28% for α-d-galactose 1-phosphate, 0% for α-d-galactosamine 1-phosphate, 100% for α-d-xylose 1-phosphate, 100% for α-d-glucosamine 1-phosphate, 70% for α-d-mannose 1-phosphate, and 0% for α-d-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity.