Computational Evidence for a Water-Assisted Proton-Transfer Mechanism in Uronate Isomerase from <i>Bacillus Halodurans</i> (Bh0493)

Uronate isomerase (EC 5.3.1.12; URI) catalyzes uronate sugar interconversion, a key step in bacterial metabolism, yet its reaction mechanism remains poorly understood. This study delineates the detailed mechanism for the isomerization of d-glucuronate to d-fructuronate catalyzed by a Zn²⁺-dependent URI enzyme (from <i>Bacillus halodurans</i>). Using quantum mechanical (QM) cluster calculations, three mechanistic pathways were evaluated, all involving Asp355 as the catalytic base but differing in the proton-shuttle mechanism. The most favorable mechanism features C5 deprotonation of the substrate, followed by a water-mediated 1,2-proton transfer via a stabilized <i>cis</i>-enediol intermediate, with the C2-C5 intramolecular transfer as the rate-determining step. The calculated activation barrier (17.3 kcal mol^{- 1}) aligns well with experimental data. Alternative pathways involving Tyr50 or a Tyr48-water relay were found to be less favorable due to higher net barriers (26-36 kcal mol^{- 1}). A comparative analysis of d-glucuronate and d-galacturonate revealed that although both proceed through similar steps, d-glucuronate has ∼2 kcal mol^{- 1} lower overall barrier due to enhanced stabilization of late-stage intermediates. These findings clarify the roles of solvent and active-site residues in URI catalysis and contribute to a broader understanding of proton-transfer mechanisms in Zn²⁺-dependent enzymes across the amidohydrolase superfamily.