Dataset Information

Substrate-dependent dynamics of UDP-galactopyranose mutase: Implications for drug design.

ABSTRACT: 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.

SUBMITTER: Boechi L

PROVIDER: S-EPMC3831665 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Substrate-dependent dynamics of UDP-galactopyranose mutase: Implications for drug design.

Boechi Leonardo L de Oliveira Cesar Augusto F CA Da Fonseca Isabel I Kizjakina Karina K Sobrado Pablo P Tanner John J JJ McCammon J Andrew JA

Protein science : a publication of the Protein Society 20130917 11

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-galac ...[more]

PMID: 23934860

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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.

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.

Dataset Information

Substrate-dependent dynamics of UDP-galactopyranose mutase: Implications for drug design.

Publications

Substrate-dependent dynamics of UDP-galactopyranose mutase: Implications for drug design.

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