Study of Uridine 5'-Diphosphate (UDP)-Galactopyranose Mutase Using UDP-5-Fluorogalactopyranose as a Probe: Incubation Results and Mechanistic Implications.
Ontology highlight
ABSTRACT:
SUBMITTER: Lin GM
PROVIDER: S-EPMC5474323 | biostudies-literature | 2016 Jul
REPOSITORIES: biostudies-literature
Publications
Study of Uridine 5'-Diphosphate (UDP)-Galactopyranose Mutase Using UDP-5-Fluorogalactopyranose as a Probe: Incubation Results and Mechanistic Implications.
Lin Geng-Min GM Sun He G HG Liu Hung-Wen HW
Organic letters 20160707 14
Uridine 5'-diphosphate-5-fluorogalactopyranose (UDP-5F-Galp, 7) was synthesized, and its effect on UDP-Galp mutase (UGM) was investigated. UGM facilitated the hydrolysis of 7 to yield UDP and 5-oxogalactose (24), but no 11 was detected. (19)F NMR and trapping experiments demonstrated that the reaction involves the initial formation of a substrate-cofactor adduct followed by decomposition of the resulting C5 gem-fluorohydrin to generate a 5-oxo intermediate (10). The results support the current m ...[more]
Similar Datasets
Project description:Nematodes represent a diverse phylum of both free living and parasitic species. While the species Caenorhabditis elegans is a valuable model organism, parasitic nematodes or helminths pose a serious threat to human health. Indeed, helminths cause many neglected tropical diseases that afflict humans. Nematode glycoconjugates have been implicated in evasive immunomodulation, a hallmark of nematode infections. One monosaccharide residue present in the glycoconjugates of several human pathogens is galactofuranose (Galf). This five-membered ring isomer of galactose has not been detected in mammals, making Galf metabolic enzymes attractive therapeutic targets. The only known pathway for biosynthetic incorporation of Galf into glycoconjugates depends upon generation of the glycosyl donor UDP-Galf by the flavoenzyme uridine 5'-diphosphate (UDP) galactopyranose mutase (UGM or Glf). A putative UGM encoding gene (glf-1) was recently identified in C. elegans. We sought to assess the catalytic activity of the corresponding gene product (CeUGM). CeUGM catalyzes the isomerization of UDP-Galf and UDP-galactopyranose (UDP-Galp). In the presence of enzyme, substrate, and a hydride source, a galactose-N5-FAD adduct was isolated, suggesting the CeUGM flavin adenine dinucleotide (FAD) cofactor serves as a nucleophile in covalent catalysis. Homology modeling and protein variants indicate that CeUGM possesses an active site similar to that of prokaryotic enzymes, despite the low sequence identity (?15%) between eukaryotic and prokaryotic UGM proteins. Even with the primary sequence differences, heterocyclic UGM inhibitors developed against prokaryotic proteins also inhibit CeUGM activity. We postulate that inhibitors of CeUGM can serve as chemical probes of Galf in nematodes and as anthelmintic leads. The available data suggest that CeUGM facilitates the biosynthetic incorporation of Galf into nematode glycoconjugates through generation of the glycosyl donor UDP-Galf.
| S-EPMC3779688 | biostudies-literature
Project description:Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that represents one of the major health challenges of the Latin American countries. Successful efforts were made during the last few decades to control the transmission of this disease, but there is still no treatment for the 10 million adults in the chronic phase of the disease. In T. cruzi, as well as in other pathogens, the flavoenzyme UDP-galactopyranose mutase (UGM) catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, a precursor of the cell surface ?-galactofuranose that is involved in the virulence of the pathogen. The fact that UGM is not present in humans makes inhibition of this enzyme a good approach in the design of new Chagas therapeutics. By performing a series of computer simulations of T. cruzi UGM in the presence or absence of an active site ligand, we address the molecular details of the mechanism that controls the uptake and retention of the substrate. The simulations suggest a modular mechanism in which each moiety of the substrate controls the flexibility of a different protein loop. Furthermore, the calculations indicate that interactions with the substrate diphosphate moiety are especially important for stabilizing the closed active site. This hypothesis is supported with kinetics measurements of site-directed mutants of T. cruzi UGM. Our results extend our knowledge of UGM dynamics and offer new alternatives for the prospective design of drugs.
| S-EPMC3831665 | biostudies-literature
Project description:UDP-galactopyranose mutase (Glf or UGM) catalyzes the formation of uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf) from UDP-galactopyranose (UDP-Galp). The enzyme is required for the production of Galf-containing glycans. UGM is absent in mammals, but members of the Corynebacterineae suborder require UGM for cell envelope biosynthesis. The need for UGM in some pathogens has prompted the search for inhibitors that could serve as antibiotic leads. Optimizing inhibitor potency, however, has been challenging. The UGM from Klebsiella pneumoniae (KpUGM), which is not required for viability, is more effectively impeded by small-molecule inhibitors than are essential UGMs from species such as Mycobacterium tuberculosis or Corynebacterium diphtheriae. Why KpUGM is more susceptible to inhibition than other orthologs is not clear. One potential source of difference is UGM ortholog conformation. We previously determined a structure of CdUGM bound to a triazolothiadiazine inhibitor in the open form, but it was unclear whether the small-molecule inhibitor bound this form or to the closed form. By varying the terminal tag (CdUGM-His6 and GSG-CdUGM), we crystallized CdUGM to capture the enzyme in different conformations. These structures reveal a pocket in the active site that can be exploited to augment inhibitor affinity. Moreover, they suggest the inhibitor binds the open form of most prokaryotic UGMs but can bind the closed form of KpUGM. This model and the structures suggest strategies for optimizing inhibitor potency by exploiting UGM conformational flexibility.
| S-EPMC5739916 | biostudies-literature
Project description:The flavoenzyme UDP-galactopyranose mutase (UGM) is a key enzyme in galactofuranose biosynthesis. The enzyme catalyzes the 6-to-5 ring contraction of UDP-galactopyranose to UDP-galactofuranose. Galactofuranose is absent in humans yet is an essential component of bacterial and fungal cell walls and a cell surface virulence factor in protozoan parasites. Thus, inhibition of galactofuranose biosynthesis is a valid strategy for developing new antimicrobials. UGM is an excellent target in this effort because the product of the UGM reaction represents the first appearance of galactofuranose in the biosynthetic pathway. The UGM reaction is redox neutral, which is atypical for flavoenzymes, motivating intense examination of the chemical mechanism and structural features that tune the flavin for its unique role in catalysis. These studies show that the flavin functions as nucleophile, forming a flavin-sugar adduct that facilitates galactose-ring opening and contraction. The 3-dimensional fold is novel and conserved among all UGMs, however the larger eukaryotic enzymes have additional secondary structure elements that lead to significant differences in quaternary structure, substrate conformation, and conformational flexibility. Here we present a comprehensive review of UGM three-dimensional structure, provide an update on recent developments in understanding the mechanism of the enzyme, and summarize computational studies of active site flexibility.
| S-EPMC3946560 | biostudies-literature
Project description:Aspergillus fumigatus is an opportunistic human pathogen responsible for deadly, invasive infections in immunocompromised patients. The A. fumigatus cell wall is a complex network of polysaccharides among them galactofuran, which is absent in humans. UDP-galactopyranose mutase (UGM) catalyzes the conversion of UDP-galactofuranose (UDP-Galf) to UDP-galactopyranose (UDP-Galp) and is an important virulence factor. UGM is a flavin-dependent enzyme that requires the reduced flavin for activity; flavin reduction is achieved by reaction with NADPH. The aim of this work was to discover inhibitors of UGM by targeting the NADPH binding site using an ADP-TAMRA probe in a high-throughput screening assay. The flavonoids (2S)-hesperetin and (2S)-naringenin were validated as competitive inhibitors of UGM against NADPH with Ki values of 6 µM and 74 µM, respectively. To gain insight into the active chemical substituents involved in the inhibition of UGM, several derivatives of these inhibitors were studied. The results show that the hydroxyl groups of (2S)-hesperetin are important for inhibition, in particular the phenyl-chroman moiety. Congo red susceptibility assay and growth temperature effects showed that these compounds affected cell wall biosynthesis in A. fumigatus. This work is the first report of inhibition studies on UGM from eukaryotic human pathogens.
| S-EPMC5589893 | biostudies-literature
Project description:Parasitic nematodes pose a serious threat to agriculture, livestock, and human health. Increasing resistance to antiparasitic agents underscores the need to replenish our anthelmintic arsenal. The nonpathogenic Caenorhabditis elegans, which serves as an effective model of parasitic helminths, has been used to search for new anthelmintic leads. We previously reported small-molecule inhibitors of the essential C. elegans protein UDP-galactopyranose mutase (UGM or Glf). This enzyme is required for the generation of galactofuranose (Galf)-containing glycans and is needed in nematodes for proper cuticle formation. Though our first-generation inhibitors were effective in vitro, they elicited no phenotypic effects. These findings are consistent with the known difficulty of targeting nematodes. C. elegans is recalcitrant to pharmacological modulation; typically, less than 0.02% of small molecules elicit a phenotypic effect, even at 40 ?M. We postulated that the lack of activity of the UGM inhibitors was due to their carboxylic acid group, which can be exploited by nematodes for detoxification. We therefore tested whether replacement of the carboxylate with an N-acylsulfonamide surrogate would result in active compounds. UGM inhibitors with the carboxylate mimetic can phenocopy the deleterious consequences of UGM depletion in C. elegans. These findings support the use of UGM inhibitors as anthelmintic agents. They also outline a strategy to render small-molecule carboxylates more effective against nematodes.
| S-EPMC5704970 | biostudies-literature
Project description:Galactofuranose (Galf) residues are present in cell wall glycoconjugates of numerous pathogenic microbes. Uridine 5'-diphosphate (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 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 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 A 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.
| S-EPMC2771219 | biostudies-literature
Project description: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 Ni(2+)-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.
| S-EPMC3325819 | biostudies-literature
Project description: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 2.25 A. The structures of Klebsiella pneumoniae mutase with FAD and with FADH- bound have been determined to 2.2 A and 2.35 A resolution, 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 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.
| S-EPMC3326527 | biostudies-literature
Project description:Uridine diphosphate galactopyranose mutase (UGM also known as Glf) is a biosynthetic enzyme required for construction of the galactan, an essential mycobacterial cell envelope polysaccharide. Our group previously identified two distinct classes of UGM inhibitors; each possesses a carboxylate moiety that is crucial for potency yet likely detrimental for cell permeability. To enhance the antimycobacterial potency, we sought to replace the carboxylate with a functional group mimic-an N-acylsulfonamide group. We therefore synthesized a series of N-acylsulfonamide analogs and tested their ability to inhibit UGM. For each inhibitor scaffold tested, the N-acylsulfonamide group functions as an effective carboxylate surrogate. Although the carboxylates and their surrogates show similar activity against UGM in a test tube, several N-acylsulfonamide derivatives more effectively block the growth of Mycobacterium smegmatis. These data suggest that the replacement of a carboxylate with an N-acylsulfonamide group could serve as a general strategy to augment antimycobacterial activity.
| S-EPMC5745143 | biostudies-literature