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