MODELING XYLANASE SUBSTRATE SPECIFICITY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Degradation of hemicellulose to constituent sugars and consequently into ethanol is a key step in biofuel production, given that hemicellulose is the second most abundant polysaccharide in the plant cell wall. Xylans are ¿1,4- or ¿1,3-linked polymers of xylopyranose that can be found decorated with various sugar branches. Xylanases (endo-¿1,4-xylanases) hydrolyze the internal glycosidic bonds of ¿1,4 xylans through a double displacement reaction with retention of the anomeric configuration of the glycone sugar. How these enzymes bind to the relatively linear structure with their V-shaped substrate binding clefts however remains to be understood completely. The aim of this project can be summarized as: Aim 1: Use molecular docking and molecular dynamics simulations, together with existing crystallographic data, to determine how the distal regions of the substrate binding cleft of Xylanase contributes to the presentation of polymeric substrates into the active site of these enzymes. Aim 2: Perform computational energy decomposition on substrate-enzyme complexes to identify regions of the substrate binding cleft that can be engineered to increase recognition of branched substrates, or to introduce novel subsites into the glycone region of Xylanases to increase catalytic efficiency. Aim 3: Perform site-directed and high-throughput saturation, mutagenesis of the key residue positions, predicted in Aims 1 and 2, and evaluate the subsequent clones for enhanced glycosidase activity and substrate specificity.