Investigation of Binding of UDP-Gal<i>f</i> and UDP-[3-F]Gal<i>f</i> to UDP-galactopyranose Mutase by STD-NMR Spectroscopy, Molecular Dynamics, and CORCEMA-ST Calculations

Yuan, Yue; Bleile, Dustin W.; Wen, Xin; Sanders, D.A.R.; Itoh, Kenji; Liu, Hung Wen; Pinto, B. Mario

doi:10.1021/ja7104152

Cited by 70 publications

(96 citation statements)

References 53 publications

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“…Interestingly, the K d value of 4 was determined by STD-NMR competition experiments to be in the low micromolar range, around 5-10 µM, depending on the oxidation state of the enzyme, while the corresponding UDP-3-deoxy-3-fluoro-D-galactofuranose 5 showed a much higher K d value (~400-600 µM). 35 In 2004, Kiessling and coworkers characterized a covalent adduct of the FAD cofactor and a radiolabeled UDP-Galp by mass spectrometry after having reduced the putative iminium intermediate 8 with sodium cyanoborohydride (Scheme 2). 8 These results strongly suggested that the three FAD-galactose covalent adducts 7-9 are intermediates of the reaction.…”

Section: Introductionmentioning

confidence: 99%

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: Introductionmentioning

confidence: 99%

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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“…As a way of overcoming those difficulties, accurate K D values have been determined by competition studies, [4,8] in which the unknown K D of a ligand is determined by monitoring its displacement from the protein binding site while a reference ligand of known affinity is titrated over the same sample, using the Cheng-Prusoff equation. [9] The applicability and robustness of this com petitive approach have been broadly demonstrated, [4,8,10] and even novel modified pulse sequences using isotopically labeled ligands have been devised to avoid possible problems of signal overlap. [11] Nevertheless, the main drawback is the need for a reference competitive inhibitor for which the affinity must be previously known by other technique.…”

Section: Introductionmentioning

confidence: 99%

Ligand–Receptor Binding Affinities from Saturation Transfer Difference (STD) NMR Spectroscopy: The Binding Isotherm of STD Initial Growth Rates

Angulo¹,

Enríquez-Navas²,

Nieto³

2010

Chemistry A European J

180

213

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The direct evaluation of dissociation constants (K(D)) from the variation of saturation transfer difference (STD) NMR spectroscopy values with the receptor-ligand ratio is not feasible due to the complex dependence of STD intensities on the spectral properties of the observed signals. Indirect evaluation, by competition experiments, allows the determination of K(D), as long as a ligand of known affinity is available for the protein under study. Herein, we present a novel protocol based on STD NMR spectroscopy for the direct measurements of receptor-ligand dissociation constants (K(D)) from single-ligand titration experiments. The influence of several experimental factors on STD values has been studied in detail, confirming the marked impact on standard determinations of protein-ligand affinities by STD NMR spectroscopy. These factors, namely, STD saturation time, ligand residence time in the complex, and the intensity of the signal, affect the accumulation of saturation in the free ligand by processes closely related to fast protein-ligand rebinding and longitudinal relaxation of the ligand signals. The proposed method avoids the dependence of the magnitudes of ligand STD signals at a given saturation time on spurious factors by constructing the binding isotherms using the initial growth rates of the STD amplification factors, in a similar way to the use of NOE growing rates to estimate cross relaxation rates for distance evaluations. Herein, it is demonstrated that the effects of these factors are cancelled out by analyzing the protein-ligand association curve using STD values at the limit of zero saturation time, when virtually no ligand rebinding or relaxation takes place. The approach is validated for two well-studied protein-ligand systems: the binding of the saccharides GlcNAc and GlcNAcbeta1,4GlcNAc (chitobiose) to the wheat germ agglutinin (WGA) lectin, and the interaction of the amino acid L-tryptophan to bovine serum albumin (BSA). In all cases, the experimental K(D) measured under different experimental conditions converged to the thermodynamic values. The proposed protocol allows accurate determinations of protein-ligand dissociation constants, extending the applicability of the STD NMR spectroscopy for affinity measurements, which is of particular relevance for those proteins for which a ligand of known affinity is not available.

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“…This arginine appears to stabilize the negatively charged diphosphate backbone of the sugar nucleotide substrate. Many synthetic analogues (21)(22)(23)(24)(25)(26) have been used to probe the mechanism of UGM and investigate substrate binding, but until recently, no ligand-bound crystal structures have been available. Tryptophan fluorescence (15) and molecular modeling have predicted that the uridine of the UDP-Gal base stacks with Trp-160 (numbering for K. pneumoniae) (27); in contrast, recent crystal structures of the K. pneumoniae UGM with bound UDP-Glcp (28) and UDP-Galp (29) show that the uridine stacks against tyrosine 155 in the active site.…”

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confidence: 99%

Characterization of a Bifunctional Pyranose-Furanose Mutase from Campylobacter jejuni 11168

Poulin

Nothaft

Hug

et al. 2010

Journal of Biological Chemistry

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UDP-galactopyranose mutases (UGM) are the enzymes responsible for the synthesis of UDP-galactofuranose (UDP-Galf) from UDP-galactopyranose (UDP-Galp). The enzyme, encoded by the glf gene, is present in bacteria, parasites, and fungi that express Galf in their glycoconjugates. Recently, a UGM homologue encoded by the cj1439 gene has been identified in Campylobacter jejuni 11168, an organism possessing no Galf-containing glycoconjugates. However, the capsular polysaccharide from this strain contains a 2-acetamido-2-deoxy-D-galactofuranose (GalfNAc) moiety. Using an in vitro high performance liquid chromatography assay and complementation studies, we characterized the activity of this UGM homologue. The enzyme, which we have renamed UDP-N-acetylgalactopyranose mutase (UNGM), has relaxed specificity and can use either UDP-Gal or UDP-GalNAc as a substrate. Complementation studies of mutase knock-outs in C. jejuni 11168 and Escherichia coli W3110, the latter containing Galf residues in its lipopolysaccharide, demonstrated that the enzyme recognizes both UDP-Gal and UDP-GalNAc in vivo. A homology model of UNGM and site-directed mutagenesis led to the identification of two active site amino acid residues involved in the recognition of the UDP-GalNAc substrate. The specificity of UNGM was characterized using a two-substrate co-incubation assay, which demonstrated, surprisingly, that UDP-Gal is a better substrate than UDP-GalNAc.In nature, hexose sugars are found predominantly in the thermodynamically favored pyranose ring form; however, hexose sugars in the furanose ring form are found in bacteria, fungi, and parasites (1, 2). For example, D-galactofuranose (Galf) 5 is a component in many microbial cell surface oligosaccharides (3,

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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

Cited by 70 publications

References 53 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

Ligand–Receptor Binding Affinities from Saturation Transfer Difference (STD) NMR Spectroscopy: The Binding Isotherm of STD Initial Growth Rates

Characterization of a Bifunctional Pyranose-Furanose Mutase from Campylobacter jejuni 11168

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