Crystallographic snapshots of UDP-glucuronic acid 4-epimerase ligand binding, rotation, and reduction

Luca Giacinto Iacovino, Simone Savino, Annika J.E. Borg, Claudia Binda, Bernd Nidetzky*, Andrea Mattevi

*Korrespondierende/r Autor/in für diese Arbeit

Publikation: Beitrag in einer FachzeitschriftArtikel

Abstract

UDP-glucuronic acid is converted to UDP-galacturonic acid en route to a variety of sugar-containing metabolites. This reaction is performed by a NAD1-dependent epimerase belonging to the short-chain dehydrogenase/reductase family. We present several high-resolution crystal structures of the UDP-glucuronic acid epimerase from Bacillus cereus. The geometry of the substrate-NAD+ interactions is finely arranged to promote hydride transfer. The exquisite complementarity between glucuronic acid and its binding site is highlighted by the observation that the unligated cavity is occupied by a cluster of ordered waters whose positions overlap the polar groups of the sugar substrate. Co-crystallization experiments led to a structure where substrate- and product-bound enzymes coexist within the same crystal. This equilibrium structure reveals the basis for a “swing and flip” rotation of the pro-chiral 4-keto-hexose-uronic acid intermediate that results from glucuronic acid oxidation, placing the C4' atom in position for receiving a hydride ion on the opposite side of the sugar ring. The product-bound active site is almost identical to that of the substrate-bound structure and satisfies all hydrogen-bonding requirements of the ligand. 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. These data highlight the double nature of the enzymatic mechanism: the active site features a high degree of precision in substrate recognition combined with the flexibility required for intermediate rotation.

Originalspracheenglisch
Seiten (von - bis)12461-12473
Seitenumfang13
FachzeitschriftJournal of Biological Chemistry
Jahrgang295
Ausgabenummer35
DOIs
PublikationsstatusVeröffentlicht - 28 Aug 2020

ASJC Scopus subject areas

  • !!Biochemistry
  • !!Molecular Biology
  • Zellbiologie

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