QM/MM studies on the calcium-assisted β-elimination mechanism of pectate lyase from bacillus subtilis

Pectate lyase utilizes the anti-β-elimination chemistry to catalyze the cleavage of α-1,4 glycosidic bond between _D -galacturonate regions during the degradation of plant polysaccharide pectin. We report here detailed mechanistic studies of the Bacillus subtilis pectate lyase (BsPel) using QM/MM calculations. It was found that the residue Arg279 serves as the catalytic base to abstract the α-proton from C₅₂ atom of substrate Ada2 subsite, forming an unstable carbanion intermediate. The glycosidic bond of this intermediate is scissile to generate the 4,5-unsaturated digalacturonate product and a negatively charged β-leaving group. Two active site residues (Lys247 and Arg279) and two Ca²⁺ ions (Ca2 and Ca3) form hydrogen-bonding and coordination interactions with C₅₂ COO^- of Ada2, respectively, which facilitate the proton abstraction and stabilize the generated carbanion intermediates. Arg284 is not the potential proton donor to saturate the leaving group. Actually, the proton source of leaving group is the solvent water molecule rather than any active site acidic residues. In addition, the calculation results suggest that careful selections of QM- and Active-regions are essential to accurately explore the enzymatic reactions. Proteins 2016; 84:1606-1615. © 2016 Wiley Periodicals, Inc.