title: A flexible loop controlling the enzymatic activity and specificity in a glycosyl hydrolase family 19 endochitinase from barley seeds (Hordeum vulgare L.).
abstract: To examine the role of the loop structure consisting of residues 70-82 (70-82 loop) localized to +3/4 subsite of the substrate binding cleft of a family GH-19 endochitinase from barley seeds, Trp72 and Trp82 were mutated, and the mutated enzymes (W72A, W82A, and W72A/W82A) were characterized. Thermal stability and specific activities toward glycol chitin and chitin hexasaccharide were significantly affected by the individual mutations. When N-acetylglucosamine hexamer was hydrolyzed by the wild type, the beta-anomer of the substrate was preferentially hydrolyzed, producing the trimer predominantly and the dimer and tetramer in lesser amounts. When the mutated enzymes were used instead of the wild type, the enzyme cleavage sites in the hexamer substrate were clearly shifted, and the beta-anomer selectivity was eliminated. The mutation effects on the enzymatic activity and stability were much more substantial in W82A than in W72A, but surprisingly the effects of the W82A/W72A double mutation were intermediate between those of the two single mutations. A molecular dynamics simulation of the wild type and the Trp-mutated enzymes indicated that the 70-82 loop becomes more flexible upon mutation and the flexibility increases in the order of W72A, W72A/W82A and W82A. We conclude that Trp72 interacts with the sugar residue but Trp82 modulates the loop flexibility, which controls the protein stability and enzymatic properties. These tryptophan residues are likely to interact with each other, resulting in the non-additivity of mutational effects.
authors.affiliation: Department of Advanced Bioscience, Kinki University, Nara, Japan. fukamizo@nara.kindai.ac.jp
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
authors.forename: Ryoh
authors.initials: R
authors.lastname: Miyake
authors.forename: Atsushi
authors.initials: A
authors.lastname: Tamura
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ohnuma
authors.forename: Karen
authors.initials: K
authors.lastname: Skriver
authors.forename: Niko V
authors.initials: NV
authors.lastname: Pursiainen
authors.forename: André H
authors.initials: AH
authors.lastname: Juffer