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Dihydropyrimidinase (DHPase) is a key enzyme in the pyrimidine pathway, the catabolic route for synthesis of β-amino acids. It catalyzes the reversible conversion of 5,6-dihydrouracil (DHU) or 5,6-dihydrothymine (DHT) to...

Glycoside hydrolases (GH) bind tightly to the sugar moiety at the glycosidic bond being hydrolyzed to stabilize its transition state conformation. We endeavored to assess the importance of glucose-binding residues in GH family 116 (GH116) β-glucosidases, which include human β-glucosylceramidase 2 (GBA2), by mutagenesis followed by kinetic characterization, X-ray crystallography, and ONIOM calculations on Thermoanaerobacterium xylanolyticum TxGH116, the structural model for GH116 enzymes. Mutations of residues that bind at the glucose C3OH and C4OH caused 27–196-fold increases in KM for p-nitrophenyl-β-D-glucoside, and significant decreases in the kcat, up to 5000-fold. At the C6OH binding residues, mutations of E777 decreased the kcat/KM by over 60,000-fold, while R786 mutants increased both the KM (40-fold) and kcat (2–4-fold). The crystal structures of R786A and R786K suggested a larger entrance to the active site could facilitate their faster rates. ONIOM binding energy calculations identified D452, H507, E777, and R786, along with the catalytic residues E441 and D593, as strong electrostatic contributors to glucose binding with predicted interaction energies > 15 kcal mol−1, consistent with the effects of the D452, H507, E777 and R786 mutations on enzyme kinetics. The relative importance of GH116 active site residues in substrate binding and catalysis identified in this work improves the prospects for the design of inhibitors for GBA2 and the engineering of GH116 enzymes for hydrolytic and synthetic applications.

Arabidopsis thaliana cell-wall invertase 1 (AtC-WIN1), a key enzyme in sucrose metabolism in plants, catalyzes the hydrolysis of sucrose into fructose and glucose. AtCWIN1 belongs to the glycoside hydrolase GH-J clan, where two carboxylate residues (Asp23 and Glu203 in AtCWIN1) are well documented as a nucleophile and an acid/base catalyst. However, details at the atomic level about the role of neighboring residues and enzyme-substrate interactions during catalysis are not fully understood. Here, quantum mechanical/molecular mechanical (QM/MM) free-energy simulations were carried out to clarify the origin of the observed decreased rates in Asp239Ala, Asp239Asn, and Asp239Phe in AtCWIN1 compared to the wild type and delineate the role of Asp239 in catalysis. The glycosylation and deglycosylation steps were considered in both wild type and mutants. Deglycosylation is predicted to be the rate-determining step in the reaction, with a calculated overall free-energy barrier of 15.9 kcal/mol, consistent with the experimental barrier (15.3 kcal/mol). During the reaction, the −1 furanosyl ring underwent a conformational change corresponding to 3 E ↔ [E 2 ] ⧧ ↔ 1 E according to the nomenclature of saccharide structures along the full catalytic reaction. Asp239 was found to stabilize not only the transition state but also the fructosyl-enzyme intermediate, which explains findings from previous structural and mutagenesis experiments. The 1-OH•••nucleophile interaction has been found to provide an important contribution to the transition state stabilization, with a contribution of ∼7 kcal/mol, and affected glycosylation more significantly than deglycosylation. This study provides molecular insights that improve the current understanding of sucrose binding and hydrolysis in members of clan GH-J, which may benefit protein engineering research. Finally, a rationale on the sucrose inhibitor configuration in chicory 1-FEH IIa, proposed a long time ago in the literature, is also provided based on the QM/MM calculations.