Abstract
Intramolecular mobility and conformational changes of flexible loops have important roles in the structural and functional integrity of proteins. The Achaetomium sp. Xz8 endo-polygalacturonase (PG8fn) of glycoside hydrolase (GH) family 28 is distinguished for its high catalytic activity (28,000 U/mg). Structure modeling indicated that PG8fn has a flexible T3 loop that folds partly above the substrate in the active site, and forms a hydrogen bond to the substrate by a highly conserved residue Asn94 in the active site cleft. Our research investigates the catalytic roles of Asn94 in T3 loop which is located above the catalytic residues on one side of the substrate. Molecular dynamics simulation performed on the mutant N94A revealed the loss of the hydrogen bond formed by the hydroxyl group at O34 of pentagalacturonic acid and the crucial ND2 of Asn94 and the consequent detachment and rotation of the substrate away from the active site, and that on N94Q caused the substrate to drift away from its place due to the longer side chain. In line with the simulations, site-directed mutagenesis at this site showed that this position is very sensitive to amino acid substitutions. Except for the altered K m values from 0.32 (wild type PG8fn) to 0.75–4.74 mg/ml, all mutants displayed remarkably lowered k cat (~3–20,000 fold) and k cat/K m (~8–187,500 fold) values and significantly increased △(△G) values (5.92–33.47 kJ/mol). Taken together, Asn94 in the GH28 T3 loop has a critical role in positioning the substrate in a correct way close to the catalytic residues.
Highlights
Pectin is a family of very complex polysaccharides that is mainly located in the middle lamella and primary cell walls of terrestrial plants, accounting for one-third of the plant dry weight [1]
The structure of PG8fn was modified through energy minimization, and the quality validation indicated that the comparative model is sufficient for further analysis
The pentagalacturonic acid docked into the substrate binding pocket (SBP) of the wild type PG8fn showed an extended conformation, representing a highly stable enzyme-substrate complex to allow the formation of productive complex and formation of the glycosyl-enzyme intermediate
Summary
Pectin is a family of very complex polysaccharides that is mainly located in the middle lamella and primary cell walls of terrestrial plants, accounting for one-third of the plant dry weight [1]. It is composed of long galacturonic acid chains with carboxyl groups and varying methyl ester. One of the most studied and widely used commercial pectinases is PG [6]. Insight to the catalytic properties of PG is an overarching aim and worthy of extensive study. The knowledge of the structure and function of PGs is of great importance to understand the catalytic mechanism
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