(69a) The Role of Carbohydrate-Aromatic Interactions in Serratia Marcescens Chitinases

Authors: 
Jana, S., University of Kentucky
Payne, C. M., University of Kentucky
Beckham, G. T., National Renewable Energy Laboratory
Sørlie, M., Norwegian University of Life Sciences
Hamre, A. G., Norwegian University of Life Sciences
Wildberger, P., Norwegian University of Life Sciences
Holen, M. M., Norwegian University of Life Sciences


The Role of Carbohydrate-Aromatic Interactions in Serratia marcescens Chitinases
Suvamay Jana1, Anne Grethe Hamre2, Patricia Wildberger2, Matilde Menkrog Holen2, Gregg T. Beckham3, Morten Sørlie2, and Christina M. Payne1, 4

1Department of Chemical and Materials Engineering and 4Center for Computational Sciences, University of

Kentucky, Lexington, USA

2Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, Ã?s, Norway

3National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO

Abstract

Microorganisms use a host of enzymes, including processive glycoside hydrolases, to hydrolytically deconstruct recalcitrant polysaccharides. Processive glycoside hydrolases closely associate with polymer chains and repeatedly cleave glycosidic linkages without dissociating from the crystalline surface and are typically the most abundant enzymes in both natural secretomes and industrial cocktails by virtue of their significant hydrolytic potential. We anticipate a molecular-level understanding of the protein-carbohydrate interactions in processive glycoside hydrolases will facilitate rational design of active sites for enhanced biomass conversion. A notable feature of the more than 130 glycoside hydrolase families is the ubiquity of aromatic residues lining the enzyme catalytic tunnels and clefts. We hypothesize these aromatic residues have uniquely defined roles according to their local environment and position relative to the substrate. The roles include tasks such as substrate chain acquisition and binding in the catalytic tunnel, which are mediated by carbohydrate-Ï? stacking interactions. Carbohydrate-aromatic interactions in the active site may also affect substrate distortion along the catalytic itinerary. We have chosen Serratia marcescens Family 18 processive chitinases ChiA and ChiB as model systems in which to investigate our hypothesis. The roles of eight aromatic residues in the catalytic active sites of two processive chitinases, Chitinase A and B, are examined. Molecular dynamics simulations coupled with free energy thermodynamic integration calculations elucidate molecular-level insights into the individual roles of the aromatic residues. These insights are directly compared to experimental characterizations of free energy of binding and degree of processive action to understand how active site topology affects function. A comparison to cellulases aids in understanding the transferability of these findings to other glycoside hydrolases.

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