3khu | pdb_00003khu

Crystal structure of human UDP-glucose dehydrogenase Glu161Gln, in complex with thiohemiacetal intermediate

spinqualitylabelsall models 3khu, resolution 2.30Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 3khu is a 6 chain structure with sequence from Human. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: EDO, EPE, UPG Related: 3itk, 2q3e, 2qg4 Gene: UGDH (HUMAN) Activity: UDP-glucose 6-dehydrogenase, with EC number 1.1.1.22 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function [UGDH_HUMAN] Involved in the biosynthesis of glycosaminoglycans; hyaluronan, chondroitin sulfate, and heparan sulfate. Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMed Biosynthesis of UDP-glucuronic acid by UDP-glucose 6-dehydrogenase (UGDH)5 occurs through four-electron oxidation of the C6 primary alcohol of UDP-glucose in two NAD+-dependent steps. The catalytic reaction of UGDH is thought to involve a Cys nucleophile that promotes formation of a thiohemiacetal enzyme intermediate in the course of the first oxidation step. The thiohemiacetal undergoes further oxidation into a thioester, and hydrolysis of the thioester completes the catalytic cycle. Herein we present crystallographic and kinetic evidence for the human form of UGDH that clarifies participation of covalent catalysis in the enzymatic mechanism. Substitution of the putative catalytic base for water attack on thioester (Glu161) by an incompetent analog (Gln161) gave a UGDH variant (E161Q) in which the hydrolysis step had become completely rate limiting so that a thioester enzyme intermediate accumulated at steady state. Using crystallization of E161Q in the presence of 5 mM UDP-glucose and 2 mM NAD+, we succeeded in intercepting a thiohemiacetal enzyme intermediate and determined its structure at 2.3 Angstrom resolution. Cys276 was covalently modified in the structure, establishing a role for it as catalytic nucelophile of the reaction. The reactive C6 of thiohemiacetal was in a position suitable to become further oxidized by hydride transfer to NAD+. The proposed catalytic mechanism of human UGDH involves Lys220 as general base for alcohol oxidation on UDP-glucose and as oxyanion stabilizer during formation and breakdown of the thiohemiacetal and thioester enzyme intermediates. Water bonded to Asp280 deprotonates Cys276 for function as aldehyde trap and also provides oxyanion stabilization. Glu161 is the Bronsted base catalytically promoting the thioester hydrolysis. Structural and kinetic evidence that the catalytic reaction of human UDP-glucose 6-dehydrogenase involves covalent thiohemiacetal and thioester enzyme intermediates.,Egger S, Chaikuad A, Klimacek M, Kavanagh KL, Oppermann U, Nidetzky B J Biol Chem. 2011 Nov 28. PMID:22123821 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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