Monday, April 19, 2010
LL Conference Facility (Hilton Clearwater Beach)
The Glycosyl Hydrolase family 12 domain from GuxA of Acidothermus cellulolyticus is one of the most thermostable known endoglucanases, having a Tm of 84.2 °C as determined by differential scanning calorimetry and Topt of 62°C. It has been demonstrated to have activity on a number of substrates including β-glucan, arabinoxylan, xylan, and xyloglucan, making it a potentially important enzyme for biomass conversion. Rosetta simulations were run on the GH12 amino acid sequence to identify potential mutations that impact the enzymes thermostability. To better understand the structural basis for its thermotolerance, the crystal structure was determined at high resolution and evaluated in regard to known parameters conferring thermotolerance. Using CHARMM, we also conducted molecular dynamics simulations at ambient conditions to build a wild type native residue contact map of the protein in solution. Melting simulations were conducted at high temperature to identify the structural features of the enzyme which are susceptible to thermal unfolding. Selected mutants improved packing in the core of the enzyme and eliminated hydrophilic pockets identified in the enzyme crystal structure and in silico screening showed significantly improved thermal stability as measured by the number of native contacts during the melting simulations. The mutants were then tested experimentally to evaluate the molecular dynamic predictions. This study explores the sequence-structure-function relationship of the thermophilic Cel12A enzyme from A. cellulolyticus and provides insight into the effectiveness of molecular dynamic modeling of thermostability.