4.6 Article

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

期刊

JOURNAL OF BIOLOGICAL CHEMISTRY
卷 295, 期 35, 页码 12461-12473

出版社

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA120.014692

关键词

SDR; short-chain dehydrogenase; reductase; epimerase; decarboxylase; catalytic intermediates; UDP-glucuronic acid; UDP-galacturonic acid; NADH; kinetic isotope effect; crystal structure; enzyme mechanism; dehydrogenase; nicotinamide adenine dinucleotide (NAD); substrate specificity

资金

  1. Federal Ministry of Science, Research and Economy (BMWFW)
  2. Federal Ministry of Traffic, Innovation, and Technology (bmvit)
  3. Styrian Business Promotion Agency SFG
  4. Standortagentur Tirol
  5. Government of Lower Austrian
  6. Business Agency Vienna through the COMET-Funding Program
  7. Austrian Science Funds (FWF) [I3247]
  8. Italian Ministry of Education, University, and Research (MIUR): Dipartimenti di Eccellenza Program (2018-2022)-Department of Biology and Biotechnology L. Spallanzani University of Pavia
  9. Austrian Science Fund (FWF) [I3247] Funding Source: Austrian Science Fund (FWF)

向作者/读者索取更多资源

UDP-glucuronic acid is converted to UDP-galacturonic acid en route to a variety of sugar-containing metabolites. This reaction is performed by a NAD(+)-dependent epimerase belonging to the short-chain dehydrogenase/reductase family. We present several high-resolution crystal structures of the UDP-glucuronic acid epimerase fromBacillus 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.

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