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

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 & 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 apo-enzyme together with 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.

Enzymatic C4‐Epimerization of UDP‐Glucuronic Acid: Precisely Steered Rotation of a Transient 4‐Keto Intermediate for an Inverted Reaction without Decarboxylation

“…As a first step in our investigation, we analyzed the catalytic conversion of UDP-GlcA into the 4-keto intermediate with MD and QM/MM metadynamics methods (see the Supporting Information for methods). MD simulations (600 ns) starting from the UDP-GlcA complex structure with BcUGAepi (enzyme from Bacillus cereus; PDB 6ZLD [5] ) showed the pyranosyl moiety in a relaxed 4 C 1 conformation throughout. The sugar hydrogen atom at C4 remains in proximity (2.6 Å distance on average) to the nicotinamide C4' atom (Figure S1).…”

“…[2,3] UDP-D-glucuronic acid (UDP-GlcA) 4epimerase (UGAepi; EC 5.1.3.6) catalyzes stereoinversion at the C4-position of UDP-GlcA to provide D-galacturonic acid for the synthesis of cell-wall polysaccharides. [4][5][6][7][8][9] UGAepi illustrates in many ways a general problem of fundamental importance in enzyme catalysis: in order to promote the reaction efficiently, the enzyme must achieve a fine balance between protein flexibility and precise substrate positioning. [6] The UGAepi reaction consists of two catalytic steps in a canonical sugar nucleotide epimerase mechanism, [3h, 10-15] such as that of UDP-galactose 4-epimerase (Figure 1): site-specific C4-oxidation of the substrate by a tightly bound NAD coenzyme; and non-stereospecific reduction of a transient UDP-4-ketohexuronic acid intermediate by the enzyme-Figure 1.…”

“…Not only is it crucial to coordinate the immediate catalytic event with other physical steps of the reaction, it is also key in the dynamic sampling of enzyme–substrate conformers that have electrostatics and internuclear distances tuned for the bond cleavage/formation [2, 3] . UDP‐ d ‐glucuronic acid (UDP‐GlcA) 4‐epimerase (UGAepi; EC 5.1.3.6) catalyzes stereoinversion at the C4‐position of UDP‐GlcA to provide d ‐galacturonic acid for the synthesis of cell‐wall polysaccharides [4–9] . UGAepi illustrates in many ways a general problem of fundamental importance in enzyme catalysis: in order to promote the reaction efficiently, the enzyme must achieve a fine balance between protein flexibility and precise substrate positioning [6] …”

Structure-function relationships in NDP-sugar active SDR enzymes: Fingerprints for functional annotation and enzyme engineering

“…However, Sun et al pointed out the importance of the positively charged R192 and the adjacent D194 (Streptomyces viridosporus UGAE), both located in the purple wall, which might interact and stabilize the negatively charged carboxylate moiety of the substrate (Sun et al, 2020). Recently, Borg et al (2020) characterized a novel UGAE from Bacillus cereus and demonstrated the importance of an hydrogen bond network composed of four residues, namely T126, S127, S128 (all yellow wall) and T178 (193 in the orange wall), in the coordination of the carboxylate substrate (Borg et al, 2020;Iacovino et al, 2020). In the CPa2E, the yellow wall contains the catalytically important Asn and Lys (personal communication).…”

Transcriptome analysis of Harumi tangor fruits: Insights into interstock-mediated fruit quality

“…HP3 flesh showed exclusive expression of the auxin-responsive protein SAUR19 ( Kathleen and Farquharson, 2014 ), zinc-binding dehydrogenase, non-specific lipid-transfer protein 3, vinorine synthase, and transferase family. HP4 flesh exclusively expressed trans -resveratrol di-O-methyltransferase and UDP-glucuronate 4-epimerase 5 ( Iacovino et al., 2020 ). These exclusively expressed genes may have affected the observed phenotypes.…”