Probing UDP-galactopyranose mutase binding pocket: A dramatic effect on substitution of the 6-position of UDP-galactofuranose

Eppe, Guillaume; Peltier, Pauline; Daniellou, Richard; Nugier‐Chauvin, Caroline; Ferrières, Vincent; Vincent, Stéphane

doi:10.1016/j.bmcl.2008.12.014

Cited by 31 publications

(19 citation statements)

References 36 publications

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“…These observations likely explain why it was found, in a previous study, that the 6-fluoro-analogue of UDP-Galf was a poor UGM substrate with a dramatic loss of binding affinity. 36 This fact was further verified with 6-deoxy analogs and UDP-L-arabinose (a UDP-Galf analogue without the 6 CH 2 OH group).…”

Section: Comparison Of the Interactions Of The Furanoside 4 With Pyramentioning

confidence: 81%

Structural Basis of Ligand Binding to UDP-Galactopyranose Mutase from Mycobacterium tuberculosis Using Substrate and Tetrafluorinated Substrate Analogues

et al. 2015

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UDP-Galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyses the reversible conversion of UDP-Galactopyranose (UDP-Galp) to UDP-Galactofuranose (UDPGalf) and plays a key role in the biosynthesis of the mycobacterial cell wall galactofuran. A soluble, active form of UGM from Mycobacterium tuberculosis (MtUGM) was obtained from a dual His 6 -MBP tagged MtUGM construct. We present the first complex structures of MtUGM with bound substrate UDP-Galp (both oxidized flavin and reduced flavin). In addition, we have determined the complex structures of MtUGM with inhibitors (UDP and the dideoxytetrafluorinated analogs of both UDP-Galp (UDP-F 4 -Galp) and UDP-Galf (UDP-F 4 -Galf)), which represent the first complex structures of UGM with an analogue in the furanose form, as well as the first structures of dideoxy-tetrafluorinated sugar analogs bound to a protein. These structures provide detailed insight into ligand recognition by MtUGM and show a similar overall binding mode as reported for other prokaryotic UGMs. The binding of the ligand induces conformational changes in the enzyme, allowing ligand binding and active site closure. In addition, the complex structure of MtUGM with UDP-F 4 -Galf reveals the first detailed insight into how the furanose moiety binds to UGM. In particular, this study confirmed that the furanoside adopts a high energy conformation ( 4 E) within the catalytic pocket. Moreover, these investigations provide structural insights to the enhanced binding of the dideoxy-tetrafluorinated sugars compared to unmodified analogs. These results will help in the design of carbohydrate mimetics and drug development, and show the enormous possibilities on the use of polyfluorination in the design of carbohydrate mimetics.

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Section: Comparison Of the Interactions Of The Furanoside 4 With Pyramentioning

confidence: 81%

Structural Basis of Ligand Binding to UDP-Galactopyranose Mutase from Mycobacterium tuberculosis Using Substrate and Tetrafluorinated Substrate Analogues

et al. 2015

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“…Also, previous studies with E. coli UGM, it was shown that UDP-[2-deoxy-2-fluoro]Gal f functions as a substrate with a k cat value 10 3 slower than the reported k cat with UDP-Gal f [28]. In contrast, in studies with glycosyltransferases, where the formation of an oxocarbenium ion is known to occur, it has been shown that substrates with fluorine at the C2 position destabilize the formation of the oxocarbenium ion preventing the reaction from occurring [48]–[51]. Therefore, if the UGM reaction proceeded by an oxocarbenium ion, UDP-[2-deoxy-2-fluoro]Gal f should act as an inhibitor instead of as a slow substrate.…”

Section: Discussionmentioning

confidence: 93%

Chemical Mechanism of UDP-Galactopyranose Mutase from Trypanosoma cruzi: A Potential Drug Target against Chagas' Disease

et al. 2012

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UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose (Gal f ). Gal f is found in several pathogenic organisms, including the parasite Trypanosoma cruzi , the causative agent of Chagas' disease. Gal f ) 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.

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“…Both distances are too long for hydrogen bonding, supporting the possibility that the role of this absolutely conserved residue is not to facilitate initial substrate binding but rather to stabilize the UDP intermediate during turnover 30,31. Alternatively, Arg280 may interact with the 6-OH of Gal p ; the significance of this functional group to UGM catalysis has recently been investigated 38,39. In the model, the distance separating the guanidinium group of Arg280 and the 6-OH of the Gal residue is 2.9 Å, suggesting that the guanidinium side chain serves as hydrogen bond donor and the 6-OH serves as an acceptor.…”

Section: Resultsmentioning

confidence: 99%

Ligand Binding and Substrate Discrimination by UDP-Galactopyranose Mutase

Gruber

Borrok

Westler

et al. 2009

Journal of Molecular Biology

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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 UDPgalactopyranose 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 enzymesubstrate 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 UGMligand 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 Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript these differences, we derived a model for the productive UGM-substrate complex that highlights interactions that can contribute to catalysis and substrate discrimination.

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Probing UDP-galactopyranose mutase binding pocket: A dramatic effect on substitution of the 6-position of UDP-galactofuranose

Cited by 31 publications

References 36 publications

Structural Basis of Ligand Binding to UDP-Galactopyranose Mutase from Mycobacterium tuberculosis Using Substrate and Tetrafluorinated Substrate Analogues

Structural Basis of Ligand Binding to UDP-Galactopyranose Mutase from Mycobacterium tuberculosis Using Substrate and Tetrafluorinated Substrate Analogues

Chemical Mechanism of UDP-Galactopyranose Mutase from Trypanosoma cruzi: A Potential Drug Target against Chagas' Disease

Ligand Binding and Substrate Discrimination by UDP-Galactopyranose Mutase

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