The Humicola grisea Cel12A enzyme structure at 1.2 Å resolution and the impact of its free cysteine residues on thermal stability

• DOI: 10.1110/ps.03220403
• Publication Date: 2003-11-19T18:42:47Z
• Authors: Mats Sandgren; Peter J. Gualfetti; Christian Paech; Sigrid Paech; Andrew Shaw; Laurie S. Gross; Mae Saldajeno; Gunnar I. Berglund; T. Alwyn Jones; Colin Mitchinson
• Keywords: Denaturation (fissile materials); Wild type

Abstract As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have extended our previous work on the structural and biochemical diversity GH homologs include most stable fungal found, Humicola grisea Cel12A. The H. enzyme was much more irreversible thermal denaturation than Trichoderma reesei enzyme. It had an apparent midpoint ( T m ) 68.7°C, 14.3°C higher T. There are additional three cysteines found in Cel12A To determine their importance for stability, constructed single mutants which these were exchanged with corresponding residues We also introduced cysteine into stability variants determined. Substitutions at any positions affected largest effect seen C206P, has 9.1°C lower that wild type. variant gave increase 3.9°C type, P201C mutation, converse destabilizing C206P mutation . help rationalize results, determined crystal structure variant, P201C. play important role this protein, although they not involved disulfide bond.