High‐resolution X‐ray structure of UDP‐galactose 4‐epimerase complexed with UDP‐phenol

@article{Thoden1996HighresolutionXS,
  title={High‐resolution X‐ray structure of UDP‐galactose 4‐epimerase complexed with UDP‐phenol},
  author={James B. Thoden and Perry Allen Frey and Hazel M. Holden},
  journal={Protein Science},
  year={1996},
  volume={5}
}
UDP‐galactose 4‐epimerase from Escherichia coli catalyzes the interconversion of UDP‐glucose and UDP‐galactose. In recent years, the enzyme has been the subject of intensive investigation due in part to its ability to facilitate nonstereospecific hydride transfer between β‐NADH and a 4‐keto hexopyranose intermediate. The first molecular model of the epimerase from E. coli was solved to 2.5 Å resolution with crystals grown in the presence of a substrate analogue. UDP‐phenol (Bauer AJ, Rayment I… Expand
Crystal structure of UDP‐galactose 4‐epimerase‐like l‐threonine dehydrogenase belonging to the intermediate short‐chain dehydrogenase‐reductase superfamily
TLDR
The crystal structure of a l‐threonine dehydrogenase from the psychrophilic bacterium Flavobacterium frigidimaris KUC‐1, which shows no sequence similarity to conventional l‐ThrDHs, was determined in the presence of NAD and a substrate analog, glycerol, and provides a clear bench mark for distinguishing GalE‐like l‐ ThrDHs from GalEs. Expand
UDP‐galactose 4‐epimerase from Kluyveromyces fragilis: existence of subunit independent functional site
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Specific modification reagents further confirmed that the cysteine residues required for catalysis and coenzyme fluorophore reside exclusively on a single subunit negating a `subunit sharing model' of its catalytic site. Expand
Human UDP-galactose 4-Epimerase
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The three-dimensional structure of human epimerase complexed with NADH and UDP-GlcNAc is described and it is shown that in the human enzyme, the structural equivalent of Tyr-299 in the E. coli protein is replaced with a cysteine residue and the active site volume is calculated to be ∼15% larger than that observed for the bacterial epimer enzyme. Expand
UDP‐galactose 4‐epimerase from Kluyveromyces  fragilis – catalytic sites of the homodimeric enzyme are functional and regulated
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Dissimilar kinetic patterns of the reconstituted enzyme after treatment with p‐chloromercuribenzoate indicated stability of the dimeric enzyme against fast association–dissociation, which could otherwise generate multiple forms of the enzyme with functional heterogeneity. Expand
The structural basis of substrate promiscuity in UDP-hexose 4-epimerase from the hyperthermophilic Eubacterium Thermotoga maritima.
TLDR
The data showed that TM0509 is a UDP-galactosugar 4-epimerase involved in d- GalE metabolism; consequently, this study provides the first detailed characterization of a hyperthermophilic GalE, and supports the notion that TMGalE might exhibit the earliest form of sugar-epimizing enzymes in the evolution of galactose metabolism. Expand
Crystallographic snapshots of UDP-glucuronic acid 4-epimerase ligand binding, rotation, and reduction
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The structure of the apoenzyme together with the kinetic isotope effect and mutagenesis experiments further outlines a few flexible loops that exist in discrete conformations, imparting structural malleability required for ligand rotation while avoiding leakage of the catalytic intermediate and/or side reactions. Expand
Determinants of Function and Substrate Specificity in Human UDP-galactose 4′-Epimerase*
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Results serve to validate the wild-type hGALE crystal structure and fully support the hypothesis that residue 307 acts as a gatekeeper mediating substrate access to the hGale active site. Expand
Crystal structure of UDP-galactose 4-epimerase from the hyperthermophilic archaeon Pyrobaculum calidifontis.
TLDR
Structural comparison revealed that the presence of an extensive hydrophobic interaction at the subunit interface is likely the main factor contributing to the hyperthermostability of the P. calidifontis enzyme. Expand
Preliminary X-ray crystallographic studies of UDP-glucose-4-epimerase from Aspergillus nidulans.
TLDR
UDP-glucose-4-epimerase from Aspergillus nidulans was overexpressed in Escherichia coli, purified via His-tag affinity chromatography and cocrystallized with UDP-galactose using the microbatch method to obtain an initial structure solution. Expand
Crystal Structure of Binary and Ternary Complexes of Archaeal UDP-galactose 4-Epimerase-like l-Threonine Dehydrogenase from Thermoplasma volcanium*
TLDR
The substrate binding model suggests that the reaction proceeds through abstraction of the β-hydroxyl hydrogen of l-threonine via direct proton transfer driven by Tyr137, the first description of the molecular basis for the substrate recognition and thermostability of a GalE-like l-ThrDH. Expand
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TLDR
The molecular structure of UDP‐galactose 4‐epimerase from Escherichia coli has now been solved to a nominal resolution of 2.5 Å. Expand
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The abortive complex model described here suggests that Ser 124 and Tyr 149 are likely to play important roles in the catalytic mechanism of the enzyme, which catalyzes the interconversion of UDP-galactose and UDP-glucose. Expand
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TLDR
The X-ray structures for epimerase complexed with NADH/ UDP, and NAD+/UDP, refined to 1.8 and 2.0 angstrom are described, finding significant structural differences in UDP binding between the oxidized and reduced forms of the protein which most likely explain the observation that uridine nucleotides bind more tightly toEpimerase/NADH than to epimerases/Nad+. Expand
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TLDR
It is concluded that NADH associated with the purified enzyme is a component of inactive, abortive complexes (E-NADH-uridine nucleotide) that contain tightly bound uridine nucleotides in place of the epimerization intermediate UDP-4-keto-alpha-D-hexoglucopyranose, which are produced in vivo in the course of bacterial growth. Expand
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TLDR
Uridine diphosphogalactose‐4‐epimerase from E. coli has been crystallized in a form suitable for a high‐resolution X‐ray crystallographic structural analysis, and the enzyme used in these experiments was produced by a new expression system and a modified purification scheme. Expand
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TLDR
It is concluded that lysine 153 plays an important role in increasing the chemical reactivity of enzyme-bound NAD+ in the uridine nucleotide-dependent conformational change associated with reductive inactivation and the catalytic activity of UDP-galactose 4-epimerase. Expand
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Abstract Background Bacterial 3 α ,20 β -hydroxysteroid dehydrogenase reversibly oxidizes the 3 α and 20 β hydroxyl groups of steroids derived from androstanes and pregnanes. It was the firstExpand
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TLDR
The distortion of NAD+ by the binding of UDP is a long-range effect that is transmitted from the substrate binding site to the coenzyme through the protein conformational change, which apparently distorts the pi-electron distribution in the nicotinamide ring and reduces the activation energy for its reduction. Expand
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TLDR
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TLDR
A new catalytic mechanism possibly common to all the enzymes belonging to the SDR family is proposed in which a tyrosine residue acts as a catalytic base and a serine residue plays a subsidiary role of stabilizing substrate binding. Expand
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