title: QM/MM studies on the calcium-assisted β-elimination mechanism of pectate lyase from bacillus subtilis.
abstract: Pectate lyase utilizes the anti-β-elimination chemistry to catalyze the cleavage of α-1,4 glycosidic bond betweenD-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 C52atom 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.
authors.affiliation: Key Laboratory of Colloid and Interface Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shandong University, Shandong, Jinan, 250100, China.
authors.forename: Guangcai
authors.initials: G
authors.lastname: Ma
authors.affiliation: Key Laboratory of Colloid and Interface Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shandong University, Shandong, Jinan, 250100, China.|College of Chemistry and Chemical Engineering, Xuzhou Institute of Technology, Xuzhou, Jiangsu, 221111, China.
authors.forename: Wenyou
authors.initials: W
authors.lastname: Zhu
authors.affiliation: Key Laboratory of Colloid and Interface Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shandong University, Shandong, Jinan, 250100, China. yongjunliu_1@sdu.edu.cn.
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Liu
mesh_terms: D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001412:Bacillus subtilis / Q000737:chemistry* / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D006860:Hydrogen Bonding; D006868:Hydrolysis; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D010368:Pectins / Q000737:chemistry* / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011522:Protons; D011789:Quantum Theory; D055672:Static Electricity; D013400:Sugar Acids / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004187:Disaccharides; D011522:Protons; D013400:Sugar Acids; D010368:Pectins; D001120:Arginine; D011133:Polysaccharide-Lyases; C023911:pectate lyase; D008239:Lysine; C090179:digalacturonic acid; D002118:Calcium
keywords: 4,5-unsaturated digalacturonate; polysaccharide lyase; reaction mechanism; theoretical study; α-1,4-glycosidic bond