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 of GH 12 homologs to include the most stable fungal GH 12 found, Humicola grisea Cel12A. The H. grisea enzyme was much more stable to irreversible thermal denaturation than the Trichoderma reesei enzyme. It had an apparent denaturation midpoint (T m ) of 68.7°C, 14.3°C higher than the T. reesei enzyme. There are an additional three cysteines found in the H. grisea Cel12A enzyme. To determine their importance for thermal stability, we constructed three H. grisea Cel12A single mutants in which these cysteines were exchanged with the corresponding residues in the T. reesei enzyme. We also introduced these cysteine residues into the T. reesei enzyme. The thermal stability of these variants was determined. Substitutions at any of the three positions affected stability, with the largest effect seen in H. grisea C206P, which has a T m 9.1°C lower than that of the wild type. The T. reesei cysteine variant that gave the largest increase in stability, with a T m 3.9°C higher than wild type, was the P201C mutation, the converse of the destabilizing C206P mutation in H. grisea. To help rationalize the results, we have determined the crystal structure of the H. grisea enzyme and of the most stable T. reesei cysteine variant, P201C. The three cysteines in H. grisea Cel12A play an important role in the thermal stability of this protein, although they are not involved in a disulfide bond.Keywords: Thermal stability; cellulase; cellulose; endoglucanase; homolog; protein crystal structure Bacterial and fungal cellulases are widely used in the detergent, textile, and food industries, and there is continuing interest in their potential use in the conversion of cellulosic biomass to fermentable sugars. Cellulases are glycoside hydrolases found in at least 12 families of this very large group of enzymes (Henrissat and Davies 2000), and consist of cellobiohydrolases (or exoglucanases, EC 3.2.1.91) and endoglucanases (EC 3.2.1.4). Glycoside hydrolase family 12 (GH 12) members hydrolyse the -1,4-glycosidic bond in cellulose via a double displacement reaction and a glycosylenzyme intermediate that results in retention of the anomeric configuration in the product (Schulein 1997;Birsan et al. 1998). Structures of GH 12 endoglucanases from both bacterial (Sulzenbacher et al. 1997;Crennell et al. 2002) and fungal (Sandgren et al. 2001(Sandgren et al. , 2003Khademi et al. 2002) sources have now been determined, and these provide the structural framework for the whole family. As part of a program to discover cellulases with improved properties, many novel GH 12 endoglucanases have been cloned and Abbreviations: CD, circular dichroism; DTNB, 5,5Ј-dithiobis-2-nitrobenzoic acid; GH, glycoside hydrolase; HPLC, high-pressure liquid chromatography; mme, mono-methyl-ether; NCS, noncrystallographic symmetry; PEG, polyethylene glycol; RMSD, root-mean-square deviation; TFA...