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Title: Conformational Control of UDP-Galactopyranose Mutase Inhibition

ORCiD logo [1];  [1];  [1]; ORCiD logo [1]
  1. Department of Chemistry, ‡Department of Biochemistry, and §Department of Bacteriology, University of Wisconsin—Madison, Madison, Wisconsin 53706, United States
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
OSTI Identifier:
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 56; Journal Issue: 30
Country of Publication:
United States

Citation Formats

Wangkanont, Kittikhun, Winton, Valerie J., Forest, Katrina T., and Kiessling, Laura L. Conformational Control of UDP-Galactopyranose Mutase Inhibition. United States: N. p., 2017. Web. doi:10.1021/acs.biochem.7b00189.
Wangkanont, Kittikhun, Winton, Valerie J., Forest, Katrina T., & Kiessling, Laura L. Conformational Control of UDP-Galactopyranose Mutase Inhibition. United States. doi:10.1021/acs.biochem.7b00189.
Wangkanont, Kittikhun, Winton, Valerie J., Forest, Katrina T., and Kiessling, Laura L. 2017. "Conformational Control of UDP-Galactopyranose Mutase Inhibition". United States. doi:10.1021/acs.biochem.7b00189.
title = {Conformational Control of UDP-Galactopyranose Mutase Inhibition},
author = {Wangkanont, Kittikhun and Winton, Valerie J. and Forest, Katrina T. and Kiessling, Laura L.},
abstractNote = {},
doi = {10.1021/acs.biochem.7b00189},
journal = {Biochemistry},
number = 30,
volume = 56,
place = {United States},
year = 2017,
month = 7
  • Galactofuranose (Galf) residues are present in cell wall glycoconjugates of numerous pathogenic microbes. Uridine 5{prime}-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 canmore » 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 {angstrom} 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.« less
  • 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 {angstrom} resolution X-ray crystal structures of the substrate-bound enzyme in oxidized and reduced forms, respectively. In the latter, C1 of the substrate is 3.6 {angstrom} from the nucleophilic flavin N5 position. This orientation is consistentmore » with covalent catalysis by flavin.« less
  • UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, which is a central reaction in galactofuranose biosynthesis. Galactofuranose has never been found in humans but is an essential building block of the cell wall and extracellular matrix of many bacteria, fungi, and protozoa. The importance of UGM for the viability of many pathogens and its absence in humans make UGM a potential drug target. Here we report the first crystal structures and small-angle x-ray scattering data for UGM from the fungus Aspergillus fumigatus, the causative agent of aspergillosis. The structures reveal that Aspergillus UGM hasmore » several extra secondary and tertiary structural elements that are not found in bacterial UGMs yet are important for substrate recognition and oligomerization. Small-angle x-ray scattering data show that Aspergillus UGM forms a tetramer in solution, which is unprecedented for UGMs. The binding of UDP or the substrate induces profound conformational changes in the enzyme. Two loops on opposite sides of the active site move toward each other by over 10 {angstrom} to cover the substrate and create a closed active site. The degree of substrate-induced conformational change exceeds that of bacterial UGMs and is a direct consequence of the unique quaternary structure of Aspergillus UGM. Galactopyranose binds at the re face of the FAD isoalloxazine with the anomeric carbon atom poised for nucleophilic attack by the FAD N5 atom. The structural data provide new insight into substrate recognition and the catalytic mechanism and thus will aid inhibitor design.« less
  • 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 inhibitormore » design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3 {angstrom} 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 k{sub cat}. 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.« less