Processivity, Substrate Positioning, and Binding: The Role of Polar Residues in a Family 18 Glycoside Hydrolase.
The enzymatic degradation of recalcitrant polysaccharides such as cellulose (β-1,4-linked glucose) and chitin (β-1,4-linked N-acetylglucosamine) by glycoside hydrolases (GHs) is of significant biological and economical importance. In nature, depolymerization is primarily accomplished by processive GHs, which remain attached to the substrate between subsequent hydrolytic reactions. Recent computational efforts have suggested that the processive ability of a GH is directly linked to the ligand binding free energy. The contribution of individual aromatic residues in the active site of these enzymes has been extensively studied. In this study, we offer the first experimental evidence confirming correlation of binding free energy and degree of processivity and evidence that polar residues are essential for maintaining processive ability. Exchanging Thr(276) with Ala in substrate binding subsite -2 in the processive ChiA of Serratia marcescens results in a decrease in both the enthalpy (2.6 and 3.8 kcal/mol) and free energy (0.5 and 2.2 kcal/mol) for the binding to the substrate (GlcNAc)6 and the inhibitor allosamidin, respectively, compared to that of the wild type. Moreover, the initial apparent processivity as measured by [(GlcNAc)2]/[GlcNAc] ratios (17.1 ± 0.4) and chitin degradation efficiency (20%) are greatly reduced for ChiA-T276A versus those of the wild type (30.1 ± 1.5 and 75%, respectively). Mutation of Arg(172) to Ala reduces the level of recognition and positioning of the substrate into the active site. Molecular dynamics simulations indicate ChiA-R172A behaves like the wild type, but the dynamics of ChiA-T276A are greatly influenced by mutation, which is reflective of their influence on processivity.
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences , P.O. Box 5003, N-1432 Ås, Norway.
Anne Grethe
AG
Hamre
Department of Chemical and Materials Engineering, University of Kentucky , Lexington, Kentucky 40506, United States.
Suvamay
S
Jana
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences , P.O. Box 5003, N-1432 Ås, Norway.
Nicole K
NK
Reppert
Department of Chemical and Materials Engineering, University of Kentucky , Lexington, Kentucky 40506, United States.
Christina M
CM
Payne
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences , P.O. Box 5003, N-1432 Ås, Norway.
Morten
M
Sørlie
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D002686:Chitin / Q000737:chemistry*; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D011485:Protein Binding; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D002686:Chitin; D002688:Chitinases; C000601656:chitinase A, Serratia marcescens