Structure and Mechanism of Human UDP-glucose 6-Dehydrogenase

Abstract

Elevated production of the matrix glycosaminoglycan hyaluronan is strongly implicated in epithelial tumor progression. Inhibition of synthesis of the hyaluronan precursor UDP-glucuronic acid (UDP-GlcUA) therefore presents an emerging target for cancer therapy. Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes, in two NAD+-dependent steps without release of intermediate aldehyde, the biosynthetic oxidation of UDP-glucose (UDP-Glc) to UDP-GlcUA. Here, we present a structural characterization of the hUGDH reaction coordinate using crystal structures of the apoenzyme and ternary complexes of the enzyme bound with UDP-Glc/NADH and UDP-GlcUA/NAD+. The quaternary structure of hUGDH is a disc-shaped trimer of homodimers whose subunits consist of two discrete α/β domains with the active site located in the interdomain cleft. Ternary complex formation is accompanied by rigid-body and restrained movement of the N-terminal NAD+ binding domain, sequestering substrate and coenzyme in their reactive positions through interdomain closure. By alternating between conformations in and out of the active site during domain motion, Tyr14, Glu161, and Glu165 participate in control of coenzyme binding and release during 2-fold oxidation. The proposed mechanism of hUGDH involves formation and breakdown of thiohemiacetal and thioester intermediates whereby Cys276 functions as the catalytic nucleophile. Stopped-flow kinetic data capture the essential deprotonation of Cys276 in the course of the first oxidation step, allowing the thiolate side chain to act as a trap of the incipient aldehyde. Because thiohemiacetal intermediate accumulates at steady state under physiological reaction conditions, hUGDH inhibition might best explore ligand binding to the NAD+ binding domain.

UDP-glucose Dehydrogenase Activity and Optimal Downstream Cellular Function Require Dynamic Reorganization at the Dimer-Dimer Subunit Interfaces

“…Crystallographic data are available for several wild-type or point mutant UGDH forms (PDB codes 3ITK (29) and 3KHU (30)), including an apo structure as well as ternary forms with each permutation of the abortive complex (NAD ϩ cofactor and UDP-glucuronate product (PDB code 2QG4); NADH cofactor and UDP-glucose substrate (PDB code 2Q3E) (29)). These structural data provide a high resolution view of the non-covalent bonding network that sustains the subunit interface in the context of the hexamer (Fig.…”

Structural Characterization of CalS8, a TDP-α-d-Glucose Dehydrogenase Involved in Calicheamicin Aminodideoxypentose Biosynthesis

“…The absence of glucose 1-phosphate moiety could be a result of disorder in the crystal or partial hydrolysis during crystallization. The remaining TMP moiety occupies essentially the same position as that of UDP-Glc in other representative ligand-bound UGDH structures such the hUGDH⅐UDP-Glc-NADH complex (PDB 2Q3E) (where hUGDH is human UGDH) (37). It is intriguing that the root mean square deviation (RMSD) between two monomers within the dimeric structures is ϳ2.5 Å, likely indicative of allosteric interaction between two monomers.…”

“…3D), consistent with the requisite covalent bond in catalysis. A water molecule also implicated in catalysis (37,44) is observed in the CalS8 complex 3.2 Å from the substrate C6Љ-OH, which hydrogen-bonds to side-chain carboxylate of Asp 280 and side-chain hydroxyl of Thr 132 (distance ϳ2.3 and 2.8 Å, respectively) (Fig. 3D).…”

“…3, B and D). Within this set, Cys 276 is the catalytic residue that forms covalent substrate-enzyme thiohemiacetal/ester intermediates in the oxidative half-reactions en route to NDP-GlcA (2,37,(42)(43)(44). Within this context, Lys 218 has been proposed to act as general base and in oxyanion stabilization, whereas Asp 280 has been implicated in Cys 276 deprotonation and oxyanion stabilization via coordination with an active site water molecule.…”

“…Within this context, Lys 218 has been proposed to act as general base and in oxyanion stabilization, whereas Asp 280 has been implicated in Cys 276 deprotonation and oxyanion stabilization via coordination with an active site water molecule. In addition, Glu 164 /Glu 168 are proposed to function as Brønsted bases in promoting the thioester hydrolysis, whereas Asn 222 and Thr 132 are believed to also help to stabilize the substrate C6Љ-oxygen negative charge over the course of the reaction (2,37,(42)(43)(44). In the CalS8 docked model, the Cys 276 side-chain thiol is 1.9 Å from the C6Љ-OH of the sugar (Fig.…”