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Abstract
Thermostable endoglucanases (EGs) capable of hydrolyzing cellulose substrate at higher temperature are of interest to industrial cellulose conversion processes. Endoglucanases from Acidothermus cellulolyticus (EGAc) and Pyrococcus horikoshii (EGPh) are functional above 80°C and show activity toward crystalline cellulose. Since no experimental three-dimensional structure of EGPh has been reported, the aim of this study was to use computational methods to model the structure of this enzyme. Since EGPh and EGAc together share a 45% amino acid sequence identity, the crystal structure of EGAc was used to serve as a template for the structural model generation of EGPh. Computational methods including homology modeling (HM) and molecular dynamics (MD) simulations are used to aid in protein engineering of EGPh. The homology model showed that the EGPh sequence folded in a (β/α) 8 barrel topology with conserved residues located close to each other near the catalytic site. Two unique amino acid insertions in the EGPh sequence have caused some difficulty for loop modeling. MD enabled further refining of the model and allowed exploration of the enzyme dynamics. The refined homology model satisfied model assessment by PROCHECK and PROSA. MD simulation of the Y264G mutant complex showed the release of the cellotetraose substrate. This result therefore suggests the loss of this tyrosine binding platform had significant influence on the interaction between the enzyme and the substrate. Thus further experimental investigation using this mutant may be useful. The EGPh sequence of archael origin contains a large amount of charged residues and the model showed that they participate cooperatively in a salt bridge network. Therefore salt bridges may have played an important role in the thermobstability of EGPh.
