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 NAD+-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.
Highlights
Nucleotide sugars are high-energy donor molecules that fulfill many roles, including contributing to cellular and tissue structural integrity [1,2,3], acting as molecular recognition markers for xenobiotic detoxification [4,5,6], and providing scaffolds for drug development [7]
This enzyme is part of the Leloir pathway and interconverts UDP-galactose into UDP-glucose [20]. It belongs to the short-chain dehydrogenase/reductases (SDRs) family, featuring the same Ser/ThrTyr-Lys catalytic triad as UDP-glucuronic acid 4-epimerase
Overall structure The crystal structure of BcUGAepi co-purified with NAD1 was initially solved at 2.2 Å resolution by molecular replacement (Table 1)
Summary
Nucleotide sugars are high-energy donor molecules that fulfill many roles, including contributing to cellular and tissue structural integrity [1,2,3], acting as molecular recognition markers for xenobiotic detoxification [4,5,6], and providing scaffolds for drug development [7]. To visualize the mode of the sugar binding in this cavity, BcUGAepi was co-crystallized in the presence of an excess of UDP-GlcA, and the structure was solved at 1.8 Å resolution (Table 1).
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