title: Subsite-specific contributions of different aromatic residues in the active site architecture of glycoside hydrolase family 12.
abstract: The active site architecture of glycoside hydrolase (GH) is a contiguous subregion of the enzyme constituted by residues clustered in the three-dimensional space, recognizing the monomeric unit of ligand through hydrogen bonds and hydrophobic interactions. Mutations of the key residues in the active site architecture of the GH12 family exerted different impacts on catalytic efficiency. Binding affinities between the aromatic amino acids and carbohydrate rings were quantitatively determined by isothermal titration calorimetry (ITC) and the quantum mechanical (QM) method, showing that the binding capacity order of Tyr>Trp>His (and Phe) was determined by their side-chain properties. The results also revealed that the binding constant of a certain residue remained unchanged when altering its location, while the catalytic efficiency changed dramatically. Increased binding affinity at a relatively distant subsite, such as the mutant of W7Y at the -4 subsite, resulted in a marked increase in the intermediate product of cellotetraose and enhanced the reactivity of endoglucanase by 144%; while tighter binding near the catalytic center, i.e. W22Y at the -2 subsite, enabled the enzyme to bind and hydrolyze smaller oligosaccharides. Clarification of the specific roles of the aromatics at different subsites may pave the way for a more rational design of GHs.
authors.affiliation: The State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, P.R. China.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Zhang
authors.affiliation: The State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, P.R. China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Wang
authors.affiliation: The State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, P.R. China.
authors.forename: Xiuyun
authors.initials: X
authors.lastname: Wu
authors.affiliation: The State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, P.R. China.
authors.forename: Shijia
authors.initials: S
authors.lastname: Liu
authors.affiliation: The State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, P.R. China.
authors.forename: Dandan
authors.initials: D
authors.lastname: Li
authors.affiliation: Key Laboratory of Colloid and Interface Chemistry, Ministry of Education, Institute of Theoretical Chemistry, Shandong University, Jinan, 250100, P.R. China.
authors.forename: Hao
authors.initials: H
authors.lastname: Xu
authors.affiliation: The State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, P.R. China.
authors.forename: Peiji
authors.initials: P
authors.lastname: Gao
authors.affiliation: The State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, P.R. China.
authors.forename: Guanjun
authors.initials: G
authors.lastname: Chen
authors.affiliation: The State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, P.R. China.
authors.forename: Lushan
authors.initials: L
authors.lastname: Wang
mesh_terms: D019943:Amino Acid Substitution; D024322:Amino Acids, Aromatic; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D020125:Mutation, Missense
chemical_list: D024322:Amino Acids, Aromatic; D001426:Bacterial Proteins; D005656:Fungal Proteins; D006026:Glycoside Hydrolases