Galf Residues Research Articles

Distinguishing Galactoside Isomers with Mass Spectrometry and Gas-Phase Infrared Spectroscopy.

Sequencing glycans is demanding due to their structural diversity. Compared to mammalian glycans, bacterial glycans pose a steeper challenge because they are constructed from a larger pool of monosaccharide building blocks, including pyranose and furanose isomers. Though mammalian glycans incorporate only the pyranose form of galactose (Galp), many pathogens, including Mycobacterium tuberculosis and Klebsiella pneumoniae, contain galactofuranose (Galf) residues in their cell envelope. Thus, glycan sequencing would benefit from methods to distinguish between pyranose and furanose isomers of different anomeric configurations. We used infrared multiple photon dissociation (IRMPD) spectroscopy with mass spectrometry (MS-IR) to differentiate between pyranose- and furanose-linked galactose residues. These targets pose a challenge for MS-IR because the saccharides lack basic groups, and galactofuranose residues are highly flexible. We postulated cationic groups that could complex through hydrogen bonding would offer a solution. Here, we present the first MS-IR analysis of hexose ammonium adducts. We compared their IR fingerprints with those of lithium adducts. We determined the diagnostic MS-IR signatures of the α- and β-anomers of galactose in furanose and pyranose forms. We also showed these signatures could be applied to disaccharides to assign galactose ring size. Our findings highlight the utility of MS-IR for analyzing the unique substructures that occur in bacterial glycans.

Catalytic Mechanism of Processive GlfT2: Transition Path Sampling Investigation of Substrate Translocation.

We applied the transition path sampling (TPS) method to study the translocation step of the catalytic mechanism of galactofuranosyl transferase 2 (GlfT2). Using TPS in the field of enzymatic reactions is still relatively rare, and we show its effectiveness on this enzymatic system. We decipher an unknown mechanism of the translocation step and, thus, provide a complete understanding of the catalytic mechanism of GlfT2 at the atomistic level. The GlfT2 enzyme is involved in the formation of the mycobacterial cell wall and transfers galactofuranose (Galf) from UDP-Galf onto a growing acceptor Galf chain. The biosynthesis of the galactan chain is accomplished in a processive manner, with the growing acceptor substrate remaining bound to GlfT2. The glycosidic bond formed by GlfT2 between the two Galf residues alternates between β-(1–6) and β-(1–5) linkages. The translocation of the growing galactan between individual additions of Galf residues is crucial for the function of GlfT2. Analysis of unbiased trajectory ensembles revealed that the translocation proceeds differently depending on the glycosidic linkage between the last two Galf residues. We also showed that the protonation state of the catalytic residue Asp372 significantly influences the translocation. Approximate transition state structures and potential energy reaction barriers of the translocation process were determined. The calculated potential reaction barriers in the range of 6–14 kcal/mol show that the translocation process is not the rate-limiting step in galactan biosynthesis.

Chlorella vulgaris α-L-arabino-α-L-rhamno-α,β-D-galactan structure and mechanisms of its anti-inflammatory and anti-remodelling effects

Microalgal exopolysaccharides (EPSs) are given great attention due to their potential biotechnology applications. Purified C. vulgaris EPS was subjected to compositional and sugar linkage analyses, and partial acid hydrolysis. Hydrolysate separation by gel chromatography afforded oligosaccharide fractions. Both, EPS and oligomers were studied by NMR spectroscopy. Data suggest very complex highly branched α-L-arabino-α-L-rhamno-α,β-D-galactan structure. Backbone repeating unit is formed by →2)-α-L-Rha (1 → 3)-α-L-Rha(1 → sequence, highly branched by long 1,6-linked α-D-Galp side chains, further branched at C2, C3 or C4 by α-L-Araf, α-D-Galf and β-D-Galf residues. α-L-Araf form longer 1,2-linked chains branched at C3, C4 or C5. Galf residues are localized as terminal units predominantly in the β configuration, while α-D-Galp and α-L-Araf may be partially O-methylated. Ex vivo biological assays showed increased interleukin-12 (IL-12) and interferon-gamma (INF-γ) levels corresponding to transforming growth factor beta (TGF-β) decrease in guinea pig model experimental asthma. These facts point to the anti-remodelling effect of Chlorella EPS and suggest its possible application in the treatment of asthma and chronic obstructive pulmonary disorder.

Monoclonal Antibody AP3 Binds Galactomannan Antigens Displayed by the Pathogens Aspergillus flavus, A. fumigatus, and A. parasiticus.

Aspergillus fumigatus and A. flavus are the fungal pathogens responsible for most cases of invasive aspergillosis (IA). Early detection of the circulating antigen galactomannan (GM) in serum allows the prompt application of effective antifungal therapy, thus improving the survival rate of IA patients. However, the use of monoclonal antibodies (mAbs) for the diagnosis of IA is often associated with false positives due to cross-reaction with bacterial polysaccharides. More specific antibodies are therefore needed. Here we describe the characterization of the Aspergillus-specific mAb AP3 (IgG1κ), including the precise identification of its corresponding antigen. The antibody was generated using A. parasiticus cell wall fragments and was shown to bind several Aspergillus species. Immunofluorescence microscopy revealed that AP3 binds a cell wall antigen, but immunoprecipitation and enzyme-linked immunosorbent assays showed that the antigen is also secreted into the culture medium. The inability of AP3 to bind the A. fumigatus galactofuranose (Galf )-deficient mutant ΔglfA confirmed that Galf residues are part of the epitope. Several lines of evidence strongly indicated that AP3 recognizes the Galf residues of O-linked glycans on Aspergillus proteins. Glycoarray analysis revealed that AP3 recognizes oligo-[β-D-Galf-1,5] sequences containing four or more residues with longer chains more efficiently. We also showed that AP3 captures GM in serum, suggesting it may be useful as a diagnostic tool for patients with IA.

Trypanosoma cruzi surface mucins are involved in the attachment to the Triatoma infestans rectal ampoule.

BackgroundTrypanosoma cruzi, the agent of Chagas disease, is a protozoan parasite transmitted to humans by blood-sucking triatomine vectors. However, and despite its utmost biological and epidemiological relevance, T. cruzi development inside the digestive tract of the insect remains a poorly understood process.Methods/Principle findingsHere we showed that Gp35/50 kDa mucins, the major surface glycoproteins from T. cruzi insect-dwelling forms, are involved in parasite attachment to the internal cuticle of the triatomine rectal ampoule, a critical step leading to its differentiation into mammal-infective forms. Experimental evidence supporting this conclusion could be summarized as follows: i) native and recombinant Gp35/50 kDa mucins directly interacted with hindgut tissues from Triatoma infestans, as assessed by indirect immunofluorescence assays; ii) transgenic epimastigotes over-expressing Gp35/50 kDa mucins on their surface coat exhibited improved attachment rates (~2–3 fold) to such tissues as compared to appropriate transgenic controls and/or wild-type counterparts; and iii) certain chemically synthesized compounds derived from Gp35/50 kDa mucins were able to specifically interfere with epimastigote attachment to the inner lining of T. infestans rectal ampoules in ex vivo binding assays, most likely by competing with or directly blocking insect receptor(s). A solvent-exposed peptide (smugS peptide) from the Gp35/50 kDa mucins protein scaffolds and a branched, Galf-containing trisaccharide (Galfβ1–4[Galpβ1–6]GlcNAcα) from their O-linked glycans were identified as main adhesion determinants for these molecules. Interestingly, exogenous addition of a synthetic Galfβ1–4[Galpβ1–6]GlcNAcα derivative or of oligosaccharides containing this structure impaired the attachment of Dm28c but not of CL Brener epimastigotes to triatomine hindgut tissues; which correlates with the presence of Galf residues on the Gp35/50 kDa mucins’ O-glycans on the former but not the latter parasite clone.Conclusion/SignificanceThese results provide novel insights into the mechanisms underlying T. cruzi-triatomine interplay, and indicate that inter-strain variations in the O-glycosylation of Gp35/50 kDa mucins may lead to differences in parasite differentiation and hence, in parasite transmissibility to the mammalian host. Most importantly, our findings point to Gp35/50 kDa mucins and/or the Galf biosynthetic pathway, which is absent in mammals and insects, as appealing targets for the development of T. cruzi transmission-blocking strategies.

Revealing the Compact Structure of Lactic Acid Bacterial Heteroexopolysaccharides by SAXS and DLS

Molecular structures of exopolysaccharides are required to understand their functions and the relationships between the structure and physical and rheological properties. Small-angle X-ray scattering and dynamic light scattering were used in conjunction with molecular modeling to characterize solution structures of three lactic acid bacterial heteroexopolysaccharides (HePS-1, HePS-2, and HePS-3). Values of radius of gyration RG, cross-sectional radius of gyration RXS, approximate length L, and hydrodynamic diameter were not directly proportional to the molar mass and indicated the HePSs adopted a compact coil-like rather than an extended conformation. Constrained molecular modeling of 15000 randomized HePS-1 conformers resulted in five best-fit structures with R factor of 3.9-4.6% revealing random coil-like structure. Φ and Ψ angle analysis of glycosidic linkages in HePS-1 structures suggests Galf residues significantly influence the conformation. Ab initio scattering modeling of HePS-2 and HePS-3 gave excellent curve fittings with χ2 of 0.43 and 0.34 for best-fit models, respectively, compatible with coil-like conformation. The findings disclose solution behavior of HePS relevant for their interactions with biomacromolecules, for example, milk proteins.

ChemInform Abstract: Chemical Insight into the Mechanism and Specificity of GlfT2, a Bifunctional Galactofuranosyltransferase from Mycobacteria

AbstractReview: 56 refs.

Chemical Insight into the Mechanism and Specificity of GlfT2, a Bifunctional Galactofuranosyltransferase from Mycobacteria

Mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce a complex cell wall structure that is essential to survival. A key component of this structure is a glycoconjugate, the mycolyl-arabinogalactan-peptidoglycan complex, which has at its core a galactan domain composed of galactofuranose (Galf) residues linked to peptidoglycan. Because galactan biosynthesis is essential for mycobacterial viability, compounds that interfere with this process are potential therapeutic agents for treating mycobacterial diseases, including tuberculosis. Galactan biosynthesis in mycobacteria involves two glycosyltransferases, GlfT1 and GlfT2, which have been the subject of increasing interest in recent years. This Synopsis summarizes efforts to characterize the mechanism and specificity of GlfT2, which is responsible for introducing the majority of the Galf residues into mycobacterial galactan.

Fidelity and Promiscuity of a Mycobacterial Glycosyltransferase.

Members of the genus Mycobacterium cause devastating human diseases, including tuberculosis. Mycobacterium tuberculosis can resist some antibiotics because of its durable and impermeable cell envelope. This barrier is assembled from saccharide building blocks not found in mammals, including galactofuranose (Galf). Within the cell envelope, Galf residues are linked together to afford an essential polysaccharide, termed the galactan. The formation of this polymer is catalyzed by the glycosyltransferase GlfT2, a processive carbohydrate polymerase, which generates a sequence-specific polysaccharide with alternating regioisomeric β(1-5) and β(1-6) Galf linkages. GlfT2 exhibits high fidelity in linkage formation, as it will terminate polymerization rather than deviate from its linkage pattern. These findings suggest that GlfT2 would prefer an acceptor with a canonical alternating β(1-5) and β(1-6) Galf sequence. To test this hypothesis, we devised a synthetic route to assemble oligosaccharides with natural and non-natural sequences. GlfT2 could elongate each of these acceptors, even those with non-natural linkage patterns. These data indicate that the glycosyltransferase is surprisingly promiscuous in its substrate preferences. However, GlfT2 did favor some substrates: it preferentially acted on those in which the lipid-bearing Galf residue was connected to the sequence by a β(1-6) glycosidic linkage. The finding that the relative positioning of the lipid and the non-reducing end of the acceptor influences substrate selectivity is consistent with a role for the lipid in acceptor binding. The data also suggest that the fidelity of GlfT2 for generating an alternating β(1-5) and β(1-6) pattern of Galf residues arises not from preferential substrate binding but during processive elongation. These observations suggest that inhibiting the action of GlfT2 will afford changes in cell wall structure.

Determination of D-galactofuranose Content of Galactomannoproteins in Aspergillus nidulans

Galactofuranose (Galf) is a component of several polysaccharides and glycoconjugates in certain species of filamentous fungi. Galf residues are frequently found in Aspergillus glycoproteins, including N-glycans and O-mannose glycans that modify many cell wall proteins and extracellular enzymes. It is known that furanoses, contained in oligosaccharides, are detected as pyranoses after hydrolysis, and that D-galactopyranose is not contained in the galactomannoproteins of Aspergillus spp. To determine the levels of D-galactofuranose in galactomannoproteins extracted from Aspergillus nidulans (A. nidulans), we measured the amount of D-galactopyranose production after galactomannoproteins hydrolysis. The method described in this manuscript allows determination of the D-galactofuranose content of galactomannoproteins in Aspergillus spp.

Comparison between DFT- and NMR-based conformational analysis of methyl galactofuranosides

Galactofuranose (Galf) residues are found in a number of microbial polysaccharides, and knowledge of their conformation is key for developing a molecular-level understanding of their biological roles. To this end, we studied 180 conformations of methyl α- and β-Galf in aqueous solution (COSMO solvation model) using density functional theory (DFT). We compare the calculated low energy conformations to those determined from the program PSEUROT using 1H NMR data. The lowest energy ring conformation for methyl α-Galf is 2E, and this conformer is also the major solution conformation obtained by NMR spectroscopy. For methyl β-Galf, 4E is the lowest energy ring conformation; however, DFT results do not agree with the solution NMR spectroscopic results. Additionally, we developed Galf-specific Karplus-like equations from these conformations.

Synthetic UDP-galactofuranose analogs reveal critical enzyme–substrate interactions in GlfT2-catalyzed mycobacterial galactan assembly

Mycobacterial cell wall galactan, composed of alternating β-(1→5) and β-(1→6) galactofuranosyl residues, is assembled by the action of two bifunctional galactofuranosyltransferases, GlfT1 and GlfT2, which use UDP-galactofuranose (UDP-Galf) as the donor substrate. Kinetic analysis of synthetic UDP-Galf analogs identified critical interactions involved in donor substrate recognition by GlfT2, a processive polymerizing glycosyltransferase. Testing of methylated UDP-Galf analogs showed the donor substrate-binding pocket is sterically crowded. Evaluation of deoxy UDP-Galf analogs revealed that the C-6 hydroxyl group is not essential for substrate activity, and that interactions with the UDP-Galf C-3 hydroxyl group orient the substrate for turnover but appears to play no role in substrate recognition, making the 3-deoxy-analog a moderate competitive inhibitor of the enzyme. Moreover, the addition of a Galf residue deoxygenated at C-5 or C-6, or an l-arabinofuranose residue, to the growing galactan chain resulted in "dead end" reaction products, which no longer act as an acceptor for the enzyme. This finding shows dual recognition of both the terminal C-5 and C-6 hydroxyl groups of the acceptor substrate are required for GlfT2 activity, which is consistent with a recent model developed based upon a crystal structure of the enzyme. These observations provide insight into specific protein-carbohydrate interactions in the GlfT2 active site and may facilitate the design of future inhibitors.

Identification of Novel Inhibitors of UDP-Galactopyranose Mutase by Structure-Based Virtual Screening.

UDP-galactopyranose mutase (UGM) is a flavo-enzyme involved in the bacterial cell wall biosynthesis. UGM catalyzes the reversible isomerization of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). UDP-Galf is the activated precursor of galactofuranose (Galf) residues that are essential components of the cell wall of certain pathogenic bacteria such as Klebsiella pneumoniae and Mycobacterium tuberculosis. Neither UGM nor Galf residues are found in humans, making Galf biosynthesis a potential drug target for developing antibacterial agents. We report the identification of novel inhibitors of UGM by in silico docking of the LeadQuest compound database against UGM from Escherichia coli. The 13 most promising inhibitors were then evaluated against K. pneumonia and M. tuberculosis UGMs by enzyme inhibition studies. Two inhibitors were identified with IC50 values of ∼1 µM and subsequently these compounds were docked into the recently solved X-ray structure of Deinococcus radiodurans UGM. The structure-activity relationships of the initial 13 compounds evaluated as inhibitors are discussed.

Fluorescence Polarization Binding Assay forAspergillus fumigatusVirulence Factor UDP-Galactopyranose Mutase

Aspergillus fumigatus is an opportunistic human pathogenic fungus responsible for deadly lung infections in immunocompromised individuals. Galactofuranose (Galf) residues are essential components of the cell wall and play an important role in A. fumigatus virulence. The flavoenzyme UDP-galactopyranose mutase (UGM) catalyzes the isomerization of UDP-galactopyranose to UDP-galactofuranose, the biosynthetic precursor of Galf. Thus, inhibitors of UGM that block the biosynthesis of Galf can lead to novel chemotherapeutics for treating A. fumigatus-related diseases. Here, we describe the synthesis of fluorescently labeled UDP analogs and the development of a fluorescence polarization (FP) binding assay for A. fumigatus UGM (AfUGM). High-affinity binding to AfUGM was only obtained with the chromophore TAMRA, linked to UDP by either 2 or 6 carbons with Kd values of 2.6 ± 0.2 μM and 3.0 ± 0.7 μM, respectively. These values were ~6 times lower than when UDP was linked to fluorescein. The FP assay was validated against several known ligands and displayed an excellent Z′ factor (0.79 ± 0.02) and good tolerance to dimethyl sulfoxide.

Monitoring Processivity and Length Control of a Carbohydrate Polymerase

Carbohydrate polymerases are abundant in nature. Although they play vital physiological roles, the molecular mechanisms that they use for the controlled assembly of polymers are largely unknown. One fundamental issue is whether an enzyme utilizes a processive or distributive mechanism for chain elongation. The shortage of mechanistic information on polysaccharide-generating glycosyltransferases became apparent when we sought to carry out investigations of GlfT2, a glycosyltransferase essential for cell wall biosynthesis in Mycobacterium tuberculosis. GlfT2 catalyzes the formation of the cell wall galactan, which is a linear polysaccharide consisting of 20-40 repeating d-galactofuranose (Galf) residues. Recombinant GlfT2 can act on synthetic acceptors to produce polymers with lengths similar to those of endogenous galactan, indicating that GlfT2 has an intrinsic ability to control polymer length. To address whether GlfT2 utilizes a processive or distributive mechanism, we developed a mass spectrometry assay. Our approach, which relies on acceptors labeled with stable isotopes, provides direct evidence that GlfT2 is a processive polymerase that maintains contact with the glycan substrate through successive monomer additions. Given this finding, we probed further the catalytic mechanism of GlfT2 to address the basis of an observed kinetic lag phase. These studies suggest that GlfT2 possesses subsites for Galf residue binding and that substrates that can fill these subsites undergo efficient processive polymerization. The presence of these subsites and the kinetic lag phase are common features of processive enzymes. We anticipate that the strategies described herein can be applied to mechanistic studies of other carbohydrate polymerization reactions.

Serodiversity of Opsonic Antibodies against Enterococcus faecalis —Glycans of the Cell Wall Revisited

In a typing system based on opsonic antibodies against carbohydrate antigens of the cell envelope, 60% of Enterococcus faecalis strains can be assigned to one of four serotypes (CPS-A to CPS-D). The structural basis for enterococcal serotypes, however, is still incompletely understood. Here we demonstrate that antibodies raised against lipoteichoic acid (LTA) from a CPS-A strain are opsonic to both CPS-A and CPS-B strains. LTA-specific antibodies also bind to LTA of CPS-C and CPS-D strains, but fail to opsonize them. From CPS-C and CPS-D strains resistant to opsonization by anti-LTA, we purified a novel diheteroglycan with a repeating unit of →6)-β-Galf-(1→3)- β-D-Glcp-(1→ with O-acetylation in position 5 and lactic acid substitution at position 3 of the Galf residue. The purified diheteroglycan, but not LTA absorbed opsonic antibodies from whole cell antiserum against E. faecalis type 2 (a CPS-C strain) and type 5 (CPS-D). Rabbit antiserum raised against purified diheteroglycan opsonized CPS-C and CPS-D strains and passive protection with diheteroglycan-specific antiserum reduced bacterial counts by 1.4 – 3.4 logs in mice infected with E. faecalis strains of the CPS-C and CPS-D serotype. Diheteroglycan-specific opsonic antibodies were absorbed by whole bacterial cells of E. faecalis FA2-2 (CPS-C) but not by its isogenic acapsular cpsI-mutant and on native PAGE purified diheteroglycan co-migrated with the gene product of the cps-locus, suggesting that it is synthesized by this locus. In summary, two polysaccharide antigens, LTA and a novel diheteroglycan, are targets of opsonic antibodies against typeable E. faecalis strains. These cell-wall associated polymers are promising candidates for active and passive vaccination and add to our armamentarium to fight this important nosocomial pathogen.

Cell Surface of Lactococcus lactis Is Covered by a Protective Polysaccharide Pellicle

In Gram-positive bacteria, the functional role of surface polysaccharides (PS) that are not of capsular nature remains poorly understood. Here, we report the presence of a novel cell wall PS pellicle on the surface of Lactococcus lactis. Spontaneous PS-negative mutants were selected using semi-liquid growth conditions, and all mutations were mapped in a single chromosomal locus coding for PS biosynthesis. PS molecules were shown to be composed of hexasaccharide phosphate repeating units that are distinct from other bacterial PS. Using complementary atomic force and transmission electron microscopy techniques, we showed that the PS layer forms an outer pellicle surrounding the cell. Notably, we found that this cell wall layer confers a protective barrier against host phagocytosis by murine macrophages. Altogether, our results suggest that the PS pellicle could represent a new cell envelope structural component of Gram-positive bacteria.

Aspergillus nidulans UDP-glucose-4-epimerase UgeA has multiple roles in wall architecture, hyphal morphogenesis, and asexual development

Aspergillus nidulans UDP-glucose-4-epimerase UgeA interconverts UDP-glucose and UDP-galactose and participates in galactose metabolism. The sugar moiety of UDP-galactose is predominantly found as galactopyranose (Galp, the six-membered ring form), which is the substrate for UDP-galactopyranose mutase (encoded by ugmA) to generate UDP-galactofuranose (Galf, the five-membered ring form) that is found in fungal walls. In A. fumigatus, Galf residues appear to be important for virulence. The A. nidulans ugeAΔ strain is viable, and has defects including wide, slow growing, highly branched hyphae and reduced conidiation that resemble the ugmAΔ strain. As for the ugmAΔ strain, ugeAΔ colonies had substantially reduced sporulation but normal spore viability. Conidia of the ugeAΔ strain could not form colonies on galactose as a sole carbon source, however they produced short, multinucleate germlings suggesting they ceased to grow from starvation. UgeA purified from an expression plasmid had a relative molecular weight of 40.6kDa, and showed in vitro UDP-glucose-4-epimerase activity. Transmission electron microscope cross-sections of wildtype, ugeAΔ, and ugmAΔ hyphae showed they had similar cytoplasmic contents but the walls of each strain were different in appearance and thickness. Both deletion strains showed increased substrate adhesion. Localization of UgeA-GFP and UgmA-GFP was cytoplasmic, and was similar on glucose and galactose. Neither gene product had a longitudinal polarized distribution. Localization of a UgmA-mRFP in a strain that resembled the ugmAΔ strain was cytoplasmic and lacked a longitudinal polarized distribution. The roles of UgeA in A. nidulans growth and morphogenesis are consistent with the importance of Galf, and are related but not identical to the roles of UgmA.

Characterization of a Bifunctional Pyranose-Furanose Mutase from Campylobacter jejuni 11168

UDP-galactopyranose mutases (UGM) are the enzymes responsible for the synthesis of UDP-galactofuranose (UDP-Galf) from UDP-galactopyranose (UDP-Galp). The enzyme, encoded by the glf gene, is present in bacteria, parasites, and fungi that express Galf in their glycoconjugates. Recently, a UGM homologue encoded by the cj1439 gene has been identified in Campylobacter jejuni 11168, an organism possessing no Galf-containing glycoconjugates. However, the capsular polysaccharide from this strain contains a 2-acetamido-2-deoxy-d-galactofuranose (GalfNAc) moiety. Using an in vitro high performance liquid chromatography assay and complementation studies, we characterized the activity of this UGM homologue. The enzyme, which we have renamed UDP-N-acetylgalactopyranose mutase (UNGM), has relaxed specificity and can use either UDP-Gal or UDP-GalNAc as a substrate. Complementation studies of mutase knock-outs in C. jejuni 11168 and Escherichia coli W3110, the latter containing Galf residues in its lipopolysaccharide, demonstrated that the enzyme recognizes both UDP-Gal and UDP-GalNAc in vivo. A homology model of UNGM and site-directed mutagenesis led to the identification of two active site amino acid residues involved in the recognition of the UDP-GalNAc substrate. The specificity of UNGM was characterized using a two-substrate co-incubation assay, which demonstrated, surprisingly, that UDP-Gal is a better substrate than UDP-GalNAc.

Structural Basis of Substrate Binding to UDP-galactopyranose Mutase: Crystal Structures in the Reduced and Oxidized State Complexed with UDP-galactopyranose and UDP

D-Galactofuranose (Galf) residues are found in the cell walls of pathogenic microbes such as Mycobacterium tuberculosis, and are essential for viability. UDP-galactopyranose mutase (UGM) is a unique flavo-enzyme that catalyzes the reversible conversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). UDP-Galf is the active precursor of Galf residues found in cell walls. Despite the wealth of biochemical/mechanistic data generated for UGM, the structural basis of substrate binding is still lacking. Here, we report the crystal structures of UGM from Deinococcus radiodurans (drUGM) in complex with its natural substrate (UDP-Galp) and UDP. Crystal structures of drUGM:UDP-Galp complexes with oxidized and reduced FAD were determined at 2.36 A and 2.50 A resolution, respectively. The substrate is buried in the active site in an unusual folded conformation and the anomeric carbon of the galactose is at a favorable distance (2.8 A) from N5 of FAD to form an FAD-galactose adduct. The mobile loops in the substrate complex structure exist in a closed conformation. The drUGM-UDP complex structure was determined at 2.55 A resolution and its overall structure is identical with that of the oxidized and reduced complexes, including the conformation of the mobile loops. Comparison with the recently reported UGM:UDP-glucose complex structure reveals key differences and the structures reported here are likely to represent the productive/active conformation of UGM. These structures provide valuable insights into substrate recognition and a basis for understanding the mechanism. These complex structures may serve as a platform for structure-guided design of inhibitors of UGM.