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1.  Structural Insight into the Unique Substrate Binding Mechanism and Flavin Redox State of UDP-galactopyranose Mutase from Aspergillus fumigatus* 
The Journal of Biological Chemistry  2012;287(14):10780-10790.
Background: UDP-galactopyranose mutase (UGM) is a critical enzyme for the proper formation of the cell wall of pathogenic microbes.
Results: The structure of UGM in complex with substrate reveals novel features of substrate binding.
Conclusion: Oxidation and reduction of the flavin cofactor causes rearrangements in the active site that affect substrate binding.
Significance: These are the first structures of UGM from a eukaryotic pathogen.
UDP-galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyzes the reversible conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). As in prokaryotic UGMs, the flavin needs to be reduced for the enzyme to be active. Here we present the first eukaryotic UGM structures from Aspergillus fumigatus (AfUGM). The structures are of UGM alone, with the substrate UDP-Galp and with the inhibitor UDP. Additionally, we report the structures of AfUGM bound to substrate with oxidized and reduced flavin. These structures provide insight into substrate recognition and structural changes observed upon substrate binding involving the mobile loops and the critical arginine residues Arg-182 and Arg-327. Comparison with prokaryotic UGM reveals that despite low sequence identity with known prokaryotic UGMs the overall fold is largely conserved. Structural differences between prokaryotic UGM and AfUGM result from inserts in AfUGM. A notable difference from prokaryotic UGMs is that AfUGM contains a third flexible loop (loop III) above the si-face of the isoalloxazine ring that changes position depending on the redox state of the flavin cofactor. This loop flipping has not been observed in prokaryotic UGMs. In addition we have determined the crystals structures and steady-state kinetic constants of the reaction catalyzed by mutants R182K, R327K, R182A, and R327A. These results support our hypothesis that Arg-182 and Arg-327 play important roles in stabilizing the position of the diphosphates of the nucleotide sugar and help to facilitate the positioning of the galactose moiety for catalysis.
PMCID: PMC3322874  PMID: 22334662
Carbohydrate-binding Protein; Cell Wall; Enzyme Mutation; Enzyme Structure; Membrane Biogenesis
2.  Fluorescence Polarization Binding Assay for Aspergillus fumigatus Virulence Factor UDP-Galactopyranose Mutase 
Enzyme Research  2011;2011:513905.
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.
PMCID: PMC3159312  PMID: 21876791
3.  Identification of the NAD(P)H Binding Site of Eukaryotic UDP-Galactopyranose Mutase 
Journal of the American Chemical Society  2012;134(43):18132-18138.
UDP-galactopyranose mutase (UGM) plays an essential role in galactofuranose biosynthesis in microorganisms by catalyzing the conversion of UDP-galactopyranose to UDP-galactofuranose. The enzyme has gained attention recently as a promising target for the design of new antifungal, antitrypanosomal, and antileishmanial agents. Here we report the first crystal structure of UGM complexed with its redox partner NAD(P)H. Kinetic protein crystallography was used to obtain structures of oxidized Aspergillus fumigatus UGM (AfUGM) complexed with NADPH and NADH, as well as reduced AfUGM after dissociation of NADP+. NAD(P)H binds with the nicotinamide near the FAD isoalloxazine and the ADP moiety extending toward the mobile 200s active site flap. The nicotinamide riboside binding site overlaps that of the substrate galactopyranose moiety, thus NADPH and substrate binding are mutually exclusive. On the other hand, the pockets for the adenine of NADPH and uracil of the substrate are distinct and separated by only 6 Å, which raises the possibility of designing novel inhibitors that bind both sites. All twelve residues that contact NADP(H) are conserved among eukaryotic UGMs. Residues that form the AMP pocket are absent in bacterial UGMs, which suggests that eukaryotic and bacterial UGMs have different NADP(H) binding sites. The structures address the longstanding question of how UGM binds NAD(P)H and provide new opportunities for drug discovery.
PMCID: PMC3493617  PMID: 23036087
4.  Expression, purification and preliminary X-ray crystallographic analysis of UDP-galactopyranose mutase from Deinococcus radiodurans  
UDP-galactopyranose mutase was crystallized using the microbatch method. The crystals diffracted to 2.36 Å resolution using synchrotron radiation.
UDP-galactopyranose mutase (UGM) catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose. A UGM–substrate complex from Deinococccus radiodurans has been expressed, purified and crystallized. Crystals were obtained by the microbatch-under-oil method at room temp­erature. The crystals diffracted to 2.36 Å resolution at the Canadian Light Source The space group was found to be P212121, with unit-cell parameters a = 134.0, b = 176.6, c = 221.6 Å. The initial structure solution was determined by molecular replacement using UGM from Mycobacterium tuberculosis (PDB code 1v0j) as a template model.
PMCID: PMC2720349  PMID: 19652355
galactofuranose; UDP-galactopyranose mutase; carbohydrates
5.  Towards the crystal structure elucidation of eukaryotic UDP-galactopyranose mutase 
UDP-galactopyranose mutase from A. fumigatus was crystallized and structure solution is in progress.
UDP-galactopyranose mutase (UGM) catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose. Eukaryotic UGMs from Aspergillus fumigatus and Leishmania major have been purified to homogeneity by means of Ni2+-affinity chromatography and crystallized. Eukaryotic UGM structure elucidation was not straightforward owing to high pseudo-symmetry, twinning and very low anomalous signal. Phasing to 2.8 Å resolution using SAD was successful for L. major UGM. However, the maps could only be improved by iterative density modification and manual model building. High pseudo-symmetry and twinning prevented correct space-group assignment and the completion of structure refinement. The structure of A. fumigatus UGM to 2.52 Å resolution was determined by molecular replacement using the incomplete 2.8 Å resolution L. major UGM model.
PMCID: PMC3325819  PMID: 22505419
UDP-galactopyranose mutase; cell-wall biosynthesis; Aspergillus fumigatus
6.  Ligand Binding and Substrate Discrimination by UDP-Galactopyranose Mutase 
Journal of molecular biology  2009;391(2):327-340.
Galactofuranose (Galf) residues are present in the cell wall glycoconjugates of numerous pathogenic microbes. UDP-galactofuranose (UDP-Galf), the biosynthetic precursor of Galf-containing glycoconjugates, is produced from UDP-galactopyranose (UDP-Galp) by the flavoenzyme UDP-galactopyranose mutase (UGM). The gene encoding UGM (glf) is essential for the viability of pathogens, including Mycobacterium tuberculosis, and this finding underscores the need to understand how UGM functions. Considerable effort has been devoted to elucidating the catalytic mechanism of UGM, but progress has been hindered by a lack of structural data for an enzyme-substrate complex. Such data could reveal not only the substrate binding interactions but how UGM can act preferentially on two very different substrates, UDP-Galp and UDP-Galf, yet avoid other structurally related UDP-sugars present in the cell. Herein, we describe the first structure of a UGM-ligand complex, which provides insight into the catalytic mechanism and the molecular basis for substrate selectivity. The structure of UGM from Klebsiella pneumoniae bound to the substrate analog UDP-glucose (UDP-Glc) was solved by X-ray crystallographic methods and refined to 2.5 Å resolution. The ligand is proximal to the cofactor, a finding that is consistent with a proposed mechanism in which the reduced flavin engages in covalent catalysis. Despite this proximity, the glucose ring of the substrate analog is positioned such that it disfavors covalent catalysis. This orientation is consistent with data indicating that UDP-Glc is not a substrate for UGM. The relative binding orientations of UDP-Galp and UDP-Glc were compared using saturation transfer difference-NMR. The results indicate that the uridine moiety occupies a similar location in both ligand complexes, and this relevant binding mode is defined by our structural data. In contrast, the orientations of the glucose and galactose sugar moieties differ. To understand the consequences of these differences, we derived a model for the productive UGM-substrate complex that highlights interactions that can contribute to catalysis and substrate discrimination.
PMCID: PMC2771219  PMID: 19500588
UDP-galactopyranose mutase; Glf; cell wall biosynthesis; mycobacteria; galactofuranose
7.  X-ray crystallography reveals a reduced substrate complex of UDP-galactopyranose mutase poised for covalent catalysis by flavin† 
Biochemistry  2009;48(39):9171-9173.
The flavoenzyme uridine 5’-diphosphate galactopyranose mutase (UGM or Glf) catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose. The latter is a key building block for cell wall construction in numerous pathogens, including Mycobacterium tuberculosis. Mechanistic studies of UGM suggested a novel role for the flavin, and we previously provided evidence that the catalytic mechanism proceeds through a covalent flavin-galactose iminium. Here, we describe 2.3 and 2.5 Å resolution X-ray crystal structures of the substrate-bound enzyme in oxidized and reduced forms, respectively. In the latter, the substrate C1 is 3.6 Å from the nucleophilic flavin N5 position. This orientation is consistent with covalent catalysis by flavin.
PMCID: PMC2785223  PMID: 19719175
8.  Aspergillus nidulans Cell Wall Composition and Function Change in Response to Hosting Several Aspergillus fumigatus UDP-Galactopyranose Mutase Activity Mutants 
PLoS ONE  2014;9(1):e85735.
Deletion or repression of Aspergillus nidulans ugmA (AnugmA), involved in galactofuranose biosynthesis, impairs growth and increases sensitivity to Caspofungin, a β-1,3-glucan synthesis antagonist. The A. fumigatus UgmA (AfUgmA) crystal structure has been determined. From that study, AfUgmA mutants with altered enzyme activity were transformed into AnugmA▵ to assess their effect on growth and wall composition in A. nidulans. The complemented (AnugmA::wild type AfugmA) strain had wild type phenotype, indicating these genes had functional homology. Consistent with in vitro studies, AfUgmA residues R182 and R327 were important for its function in vivo, with even conservative amino (RK) substitutions producing AnugmA? phenotype strains. Similarly, the conserved AfUgmA loop III histidine (H63) was important for Galf generation: the H63N strain had a partially rescued phenotype compared to AnugmA▵. Collectively, A. nidulans strains that hosted mutated AfUgmA constructs with low enzyme activity showed increased hyphal surface adhesion as assessed by binding fluorescent latex beads. Consistent with previous qPCR results, immunofluorescence and ELISA indicated that AnugmA▵ and AfugmA-mutated A. nidulans strains had increased α-glucan and decreased β-glucan in their cell walls compared to wild type and AfugmA-complemented strains. Like the AnugmA▵ strain, A. nidulans strains containing mutated AfugmA showed increased sensitivity to antifungal drugs, particularly Caspofungin. Reduced β-glucan content was correlated with increased Caspofungin sensitivity. Aspergillus nidulans wall Galf, α-glucan, and β-glucan content was correlated in A. nidulans hyphal walls, suggesting dynamic coordination between cell wall synthesis and cell wall integrity.
PMCID: PMC3893270  PMID: 24454924
9.  Targeting UDP-Galactopyranose Mutases from Eukaryotic Human Pathogens 
Current Pharmaceutical Design  2013;19(14):2561-2573.
UDP-Galactopyranose mutase (UGM) is a unique flavin-dependent enzyme that catalyzes the conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). The product of this reaction is the precursor to Galf, a major component of the cell wall and of cell surface glycoproteins and glycolipids in many eukaryotic and prokaryotic human pathogens. The function of UGM is important in the virulence of fungi, parasites, and bacteria. Its role in virulence and its absence in humans suggest that UGM is an ideal drug target. Significant structural and mechanistic information has been accumulated on the prokaryotic UGMs; however, in the past few years the research interest has shifted to UGMs from eukaryotic human pathogens such as fungi and protozoan parasites. It has become clear that UGMs from prokaryotic and eukaryotic organisms have different structural and mechanistic features. The amino acid sequence identity between these two classes of enzymes is low, resulting in differences in oligomeric states, substrate binding, active site flexibility, and interaction with redox partners. However, the unique role of the flavin cofactor in catalysis is conserved among this enzyme family. In this review, recent findings on eukaryotic UGMs are discussed and presented in comparison with prokaryotic UGMs.
PMCID: PMC3624792  PMID: 23116395
UDP-galactopyranose mutase; enzyme drug target; galactofuranose; non-redox reaction; flavoenzyme; galactopyranose; inhibitors; eukaryotic pathogens.
10.  Purification, crystallization and preliminary X-ray diffraction studies of N-acetylglucosamine-phosphate mutase from Candida albicans  
Preliminary X-ray diffraction studies on N-acetylglucosamine-phosphate mutase from C. albicans are reported.
N-acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc) in eukaryotes and belongs to the α-d-phosphohexomutase superfamily. AGM1 from Candida albicans (CaAGM1) was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals obtained belong to the primitive monoclinic space group P21, with unit-cell parameters a = 60.2, b = 130.2, c = 78.0 Å, β = 106.7°. The crystals diffract X-rays to beyond 1.8 Å resolution using synchrotron radiation.
PMCID: PMC2222579  PMID: 16582501
N-acetylglucosamine-phosphate mutase; Candida albicans
11.  Quantifying the Importance of Galactofuranose in Aspergillus nidulans Hyphal Wall Surface Organization by Atomic Force Microscopy ▿ † 
Eukaryotic Cell  2011;10(5):646-653.
The fungal wall mediates cell-environment interactions. Galactofuranose (Galf), the five-member ring form of galactose, has a relatively low abundance in Aspergillus walls yet is important for fungal growth and fitness. Aspergillus nidulans strains deleted for Galf biosynthesis enzymes UgeA (UDP-glucose-4-epimerase) and UgmA (UDP-galactopyranose mutase) lacked immunolocalizable Galf, had growth and sporulation defects, and had abnormal wall architecture. We used atomic force microscopy and force spectroscopy to image and quantify cell wall viscoelasticity and surface adhesion of ugeAΔ and ugmAΔ strains. We compared the results for ugeAΔ and ugmAΔ strains with the results for a wild-type strain (AAE1) and the ugeB deletion strain, which has wild-type growth and sporulation. Our results suggest that UgeA and UgmA are important for cell wall surface subunit organization and wall viscoelasticity. The ugeAΔ and ugmAΔ strains had significantly larger surface subunits and lower cell wall viscoelastic moduli than those of AAE1 or ugeBΔ hyphae. Double deletion strains (ugeAΔ ugeBΔ and ugeAΔ ugmAΔ) had more-disorganized surface subunits than single deletion strains. Changes in wall surface structure correlated with changes in its viscoelastic modulus for both fixed and living hyphae. Wild-type walls had the largest viscoelastic modulus, while the walls of the double deletion strains had the smallest. The ugmAΔ strain and particularly the ugeAΔ ugmAΔ double deletion strain were more adhesive to hydrophilic surfaces than the wild type, consistent with changes in wall viscoelasticity and surface organization. We propose that Galf is necessary for full maturation of A. nidulans walls during hyphal extension.
PMCID: PMC3127658  PMID: 21335527
12.  Synthesis and Analysis of Substrate Analogues for UDP-Galactopyranose Mutase: Implication for an Oxocarbenium Ion Intermediate in the Catalytic Mechanism 
Organic letters  2007;9(5):879-882.
UDP-D-galactofuranose (2), which is essential for both cell growth and virulence in many pathogenic microorganisms, is converted from UDP-D-galactopyranose (UDP-Galp, 1) by the flavin adenine dinucleotide (FAD)-dependent enzyme UDP-galactopyranose mutase (UGM). Here, we report the synthesis of UDP-GalOH (13) and show it as an inhibitor for UGM with a binding affinity similar to that of 1. These results are more consistent with a mechanism involving an oxocarbenium ion intermediate in UGM catalysis.
PMCID: PMC2515276  PMID: 17266324
13.  Investigation of binding of UDP-Galf and UDP-[3-F]Galf to UDP-galactopyranose mutase by STD-NMR spectroscopy, molecular dynamics, and CORCEMA-ST calculations 
UDP-galactopyranose mutase (UGM) is the key enzyme involved in the biosynthesis of Galf. UDP-Galp and UDP-Galf are two natural substrates of UGM. A protocol that combines the use of STD-NMR spectroscopy, molecular modeling, and CORCEMA-ST calculations was applied to the investigation of the binding of UDP-Galf and its C3-fluorinated analogue to UGM from Klebsiella pneumoniae. UDP-Galf and UDP-[3-F]Galf were bound to UGM in a similar manner as UDP-Galp. The interconversions of UDP-Galf and UDP-[3-F]Galf to their galactopyranose counterparts were catalyzed by the reduced (active) UGM with different catalytic efficiencies, as observed by NMR spectroscopy. The binding affinities of UDP-Galf and UDP-[3-F]Galf were also compared with those of UDP-Galp and UDP by competition STD-NMR experiments. When UGM was in the oxidized (inactive) state, the binding affinities of UDP-Galf, UDP-Galp, and UDP-[3-F]Galf were of similar magnitudes, and were lower than that of UDP. However, when UGM was in the reduced state, UDP-Galp had higher binding affinity compared with UDP. Molecular dynamics (MD) simulations indicated that the “open” mobile loop in UGM “closes” upon binding of the substrates. Combined MD simulations and STD-NMR experiments were used to create models of UGM with UDP-Galf and UDP-[3-F]Galf as bound ligands. Calculated values of saturation-transfer effects with CORCEMA-ST (complete relaxation and conformational exchange matrix analysis of saturation transfer) were compared to the experimental STD effects, and permitted differentiation between two main conformational families of the bound ligands. Taken together, these results are used to rationalize the different rates of catalytic turnover of UDP-Galf and UDP-[3-F]Galf, and shed light on the mechanism of action of UGM.
PMCID: PMC2788240  PMID: 18278916
UDP-galactopyranose mutase; UDP-galactopyranose; UDP-galactofuranose; UDP-[3-F]galactofuranose; STD-NMR; CORCEMA-ST; GROMACS; AutoDock 3.0.5
14.  Contribution of Galactofuranose to the Virulence of the Opportunistic Pathogen Aspergillus fumigatus▿  
Eukaryotic Cell  2008;7(8):1268-1277.
The filamentous fungus Aspergillus fumigatus is responsible for a lethal disease called invasive aspergillosis that affects immunocompromised patients. This disease, like other human fungal diseases, is generally treated by compounds targeting the primary fungal cell membrane sterol. Recently, glucan synthesis inhibitors were added to the limited antifungal arsenal and encouraged the search for novel targets in cell wall biosynthesis. Although galactomannan is a major component of the A. fumigatus cell wall and extracellular matrix, the biosynthesis and role of galactomannan are currently unknown. By a targeted gene deletion approach, we demonstrate that UDP-galactopyranose mutase, a key enzyme of galactofuranose metabolism, controls the biosynthesis of galactomannan and galactofuranose containing glycoconjugates. The glfA deletion mutant generated in this study is devoid of galactofuranose and displays attenuated virulence in a low-dose mouse model of invasive aspergillosis that likely reflects the impaired growth of the mutant at mammalian body temperature. Furthermore, the absence of galactofuranose results in a thinner cell wall that correlates with an increased susceptibility to several antifungal agents. The UDP-galactopyranose mutase thus appears to be an appealing adjunct therapeutic target in combination with other drugs against A. fumigatus. Its absence from mammalian cells indeed offers a considerable advantage to achieve therapeutic selectivity.
PMCID: PMC2519766  PMID: 18552284
15.  Crystal Structures of Trypanosoma cruzi UDP-Galactopyranose Mutase Implicate Flexibility of the Histidine Loop in Enzyme Activation 
Biochemistry  2012;51(24):4968-4979.
Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitor design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3-Å movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45 % identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10–50, primarily by decreasing kcat. Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens.
PMCID: PMC3426654  PMID: 22646091
16.  Chemical Mechanism of UDP-Galactopyranose Mutase from Trypanosoma cruzi: A Potential Drug Target against Chagas' Disease 
PLoS ONE  2012;7(3):e32918.
UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose (Galf). Galf is found in several pathogenic organisms, including the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Galf) is important for virulence and is not present in humans, making its biosynthetic pathway an attractive target for the development of new drugs against T. cruzi. Although UGMs catalyze a non-redox reaction, the flavin must be in the reduced state for activity and the exact role of the flavin in this reaction is controversial. The kinetic and chemical mechanism of TcUGM was probed using steady state kinetics, trapping of reaction intermediates, rapid reaction kinetics, and fluorescence anisotropy. It was shown for the first time that NADPH is an effective redox partner of TcUGM. The substrate, UDP-galactopyranose, protects the enzyme from reacting with molecular oxygen allowing TcUGM to turnover ∼1000 times for every NADPH oxidized. Spectral changes consistent with a flavin iminium ion, without the formation of a flavin semiquinone, were observed under rapid reaction conditions. These data support the proposal of the flavin acting as a nucleophile. In support of this role, a flavin-galactose adduct was isolated and characterized. A detailed kinetic and chemical mechanism for the unique non-redox reaction of UGM is presented.
PMCID: PMC3308961  PMID: 22448231
17.  Potent Ligands for Prokaryotic UDP-Galactopyranose Mutase That Exploit an Enzyme Subsite 
Organic letters  2009;11(1):193-196.
UDP-Galactopyranose mutase (UGM or Glf), which catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose, is implicated in the viability and virulence of multiple pathogenic microorganisms. Here we report the synthesis of high-affinity ligands for UGM homologs from Klebsiella pneumoniae and Mycobacterium tuberculosis. The potency of these compounds stems from their ability to access both the substrate binding pocket and an adjacent site.
PMCID: PMC3010353  PMID: 19067595
18.  Isolation and characterization of functional Leishmania major virulence factor UDP-galactopyranose mutase 
Human parasitic pathogens of the genus Leishmania are the causative agents of cutaneous, mucocutaneous, and visceral leishmaniasis. Currently, there are millions of people infected with these diseases and over 50,000 deaths occur annually. Recently, it was shown that the flavin-dependent enzyme UDP-galactopyranose mutase (UGM) is a virulence factor in Leishmania major. UGM catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose. The product, UDP-galactofuranose, is the only source of galactofuranose which is present on the cell surface of this parasite and has been implicated to be important for host-parasite interactions. The recombinant form of this enzyme was obtained in a soluble and active form. The enzyme was shown to be active only in the reduced sate. A kcat value of 5 ± 0.2s−1 and a KM value of 87 ± 11 μM were determined with UDP galactofuranose as substrate. Different from the dimeric bacterial and tetrameric fungal UGMs, this parasitic enzyme functions as a monomer.
PMCID: PMC3086707  PMID: 21419104
UDP-galactopyranose mutase; galactofuranose; leishmaniasis; flavoenzymes; non-redox reaction
19.  Genetic and structural validation of Aspergillus fumigatus N-acetylphosphoglucosamine mutase as an antifungal target 
Bioscience Reports  2013;33(5):e00063.
Aspergillus fumigatus is the causative agent of IA (invasive aspergillosis) in immunocompromised patients. It possesses a cell wall composed of chitin, glucan and galactomannan, polymeric carbohydrates synthesized by processive glycosyltransferases from intracellular sugar nucleotide donors. Here we demonstrate that A. fumigatus possesses an active AfAGM1 (A. fumigatus N-acetylphosphoglucosamine mutase), a key enzyme in the biosynthesis of UDP (uridine diphosphate)–GlcNAc (N-acetylglucosamine), the nucleotide sugar donor for chitin synthesis. A conditional agm1 mutant revealed the gene to be essential. Reduced expression of agm1 resulted in retarded cell growth and altered cell wall ultrastructure and composition. The crystal structure of AfAGM1 revealed an amino acid change in the active site compared with the human enzyme, which could be exploitable in the design of selective inhibitors. AfAGM1 inhibitors were discovered by high-throughput screening, inhibiting the enzyme with IC50s in the low μM range. Together, these data provide a platform for the future development of AfAGM1 inhibitors with antifungal activity.
PMCID: PMC3763426  PMID: 23844980
cell wall; drug target; enzyme; inhibitor; nucleotide sugar; protein structure; AfAGM1, A. fumigatus N-acetylphosphoglucosamine mutase; AGM1, N-acetylphosphoglucosamine mutase; CaAGM1, Candida albicans AGM1; Fru-6P, fructose 6-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; GlcNAc, N-acetylglucosamine; GlcNAc-1P, N-acetylglucosamine-1-phosphate; GlcN-6P, glucosamine 6-phosphate; GFA1, glutamine: Fru-6P amidotransferase; GNA1, GlcN-6P acetyltransferase; IA, invasive aspergillosis; MIC, minimum inhibitory concentration; MM, minimal medium; RMSD, root mean square deviation; UAP1, UDP–GlcNAc pyrophosphorylase; UDP, uridine diphosphate
20.  Purification, crystallization and preliminary X-ray diffraction studies of UDP-N-acetylglucosamine pyrophosphorylase from Candida albicans  
UDP-N-acetylglucosamine pyrophosphorylase was purified and crystallized and X-ray diffraction data were collected to 2.3 Å resolution.
UDP-N-acetylglucosamine pyrophosphorylase (UAP) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine. UAP from Candida albicans was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals of the substrate and product complexes both diffract X-rays to beyond 2.3 Å resolution using synchrotron radiation. The crystals of the substrate complex belong to the triclinic space group P1, with unit-cell parameters a = 47.77, b = 62.89, c = 90.60 Å, α = 90.01, β = 97.72, γ = 92.88°, whereas those of the product complex belong to the orthorhombic space group P212121, with unit-cell parameters a = 61.95, b = 90.87, c = 94.88 Å.
PMCID: PMC2225379  PMID: 17142897
UDP-N-acetylglucosamine pyrophosphorylase; Candida albicans
21.  A novel noncovalent complex of chorismate mutase and DAHP synthase from Mycobacterium tuberculosis: protein purification, crystallization and X-ray diffraction analysis 
Two shikimate-pathway enzymes from M. tuberculosis, the intracellular chorismate mutase (MtCM) and DAHP synthase (MtDS), were produced recombinantly and purified. MtCM was crystallized alone and in complex with MtDS and analyzed by X-ray diffraction.
Chorismate mutase catalyzes a key step in the shikimate-biosynthetic pathway and hence is an essential enzyme in bacteria, plants and fungi. Mycobacterium tuberculosis contains two chorismate mutases, a secreted and an intracellular one, the latter of which (MtCM; Rv0948c; 90 amino-acid residues; 10 kDa) is the subject of this work. Here are reported the gene expression, purification and crystallization of MtCM alone and of its complex with another shikimate-pathway enzyme, DAHP synthase (MtDS; Rv2178c; 472 amino-acid residues; 52 kDa), which has been shown to enhance the catalytic efficiency of MtCM. The MtCM–MtDS complex represents the first noncovalent enzyme complex from the common shikimate pathway to be structurally characterized. Soaking experiments with a transition-state analogue are also reported. The crystals of MtCM and the MtCM–MtDS complex diffracted to 1.6 and 2.1 Å resolution, respectively.
PMCID: PMC2765898  PMID: 19851019
AroQ chorismate mutase; drug targets; enzyme catalysis; multi-enzyme complexes; Mycobacterium tuberculosis Rv0948c; noncovalent protein complexes; shikimate pathway; transition-state analogues
22.  Crystallization and preliminary X-ray crystallographic studies of Mycobacterium tuberculosis chorismate mutase 
Chorismate mutase from M. tuberculosis has been crystallized. Preliminary X-ray crystallographic studies reveal the occurrence of a dimeric molecule in the crystal asymmetric unit.
Chorismate mutase catalyzes the first committed step in the biosynthesis of the aromatic amino acids phenylalanine and tyrosine in bacteria, fungi and higher plants. The recent re-annotation of the Mycobacterium tuberculosis genome has revealed the presence of a duplicate set of genes coding for chorismate mutase. The mycobacterial gene Rv1885c bears <20% sequence homology to other bacterial chorismate mutases, thus serving as a potential target for the development of inhibitors specific to the pathogen. The M. tuberculosis chorismate mutase was crystallized in space group C2 and the crystals diffracted to a resolution of 2.2 Å. Matthews coefficient and self-rotation function calculations revealed the presence of two monomers in the asymmetric unit.
PMCID: PMC1952302  PMID: 16511071
chorismate mutase; Mycobacterium tuberculosis; dimer
23.  Crystal structures of Mycobacteria tuberculosis and Klebsiella pneumoniae UDP-galactopyranose mutase in the oxidised state and Klebsiella pneumoniae UDP-galactopyranose mutase in the (active) reduced state 
Journal of molecular biology  2005;348(4):971-982.
Uridine diphosphogalactofuranose (UDP-Galf) is the precursor of the D-galactofuranose sugar found in bacterial and parasitic cell walls, including those of many pathogens. UDP-Galf is made from UDP-galactopyranose by the enzyme UDP-galactopyranose mutase. The enzyme requires the reduced FADH− co-factor for activity. The structure of the Mycobacterium tuberculosis mutase with FAD has been determined to 2.25Å. The structures of Klebsiella pneumoniae mutase with FAD and with FADH− bound have been determined to 2.2Å and 2.35Å resolutions respectively. This is the first report of the FADH− containing structure. Two flavin dependent mechanisms for the enzyme have been proposed, one which involves a covalent adduct being formed at the flavin and the other based on electron transfer. Using our structural data, we have examined the two mechanisms. The electron transfer mechanism is consistent with the structural data, not surprisingly since it makes fewer demands on the precise positioning of atoms. A model based on a covalent adduct FAD requires repositioning of the enzyme active site and would appear to require that the isoalloxazine ring of FADH− to buckle in a particular way. However, the FADH− structure reveals that the isoalloxazine ring buckles in the opposite sense, this apparently requires the covalent adduct to trigger profound conformational changes in the protein or to buckle the FADH− opposite to that seen in the apo structure.
PMCID: PMC3326527  PMID: 15843027
Crystal structure; mutase; TB; contractase
24.  Cloning, expression, purification, crystallization and preliminary crystallographic studies of BceC, a UDP-glucose dehydrogenase from Burkholderia cepacia IST408 
Crystals of B. cepacia IST408 UDP-glucose dehydrogenase (EC were obtained in space group P212121 and diffracted to 2.09 Å resolution.
Bacteria of the Burkholderia cepacia complex (Bcc) have emerged as important opportunistic pathogens, establishing lung infections in immunocompromised or cystic fibrosis patients. Bcc uses polysaccharide-biofilm production in order to evade the host immune response. The biofilm precursor UDP-glucuronic acid is produced by a twofold NAD+-dependent oxidation of UDP-glucose. In B. cepacia IST408 this enzymatic reaction is performed by the UDP-glucose dehydrogenase BceC, a 470-residue enzyme, the production and crystallization of which are described here. The crystals belonged to the orthorhombic space group P212121 and contained four molecules in the asymmetric unit. Their crystallo­graphic analysis at 2.09 Å resolution and a molecular-replacement study are reported.
PMCID: PMC2833033  PMID: 20208157
UDP-glucose dehydrogenases; Burkholderia cepacia; BceC
25.  Synthetic UDP-furanoses as potent inhibitors of mycobacterial galactan biogenesis 
Chemistry & biology  2010;17(12):1356-1366.
UDP-galactofuranose (UDP-Galf) is a substrate for two types of enzymes, UDP-galactopyranose mutase and galactofuranosyltransferases, which are present in many pathogenic organisms but absent from mammals. In particular, these enzymes are involved in the biosynthesis of cell wall galactan, a polymer essential for the survival of the causative agent of tuberculosis, Mycobacterium tuberculosis. We describe here the synthesis of derivatives of UDP-Galf modified at C-5 and C-6 using a chemoenzymatic route. In cell-free assays, these compounds prevented the formation of mycobacterial galactan, via the production of short “dead-end” intermediates resulting from their incorporation into the growing oligosaccharide chain. Modified UDP-furanoses thus constitute novel probes for the study of the two classes of enzymes involved in mycobacterial galactan assembly, and studies with these compounds may ultimately facilitate the future development of new therapeutic agents against tuberculosis.
PMCID: PMC3012269  PMID: 21168771

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