The X-ray crystal structure of the Trichoderma reesei family 12 endoglucanase 3, Cel12A, at 1.9 Å resolution

We present the three-dimensional structure of Trichoderma reesei endoglucanase 3 (Cell2A), a small, 218 amino acid residue (24.5 kDa), neutral pi, glycoside hydrolase family 12 cellulase that lacks a cellulose-binding module. The structure has been determined using X-ray crystallography and refined to 1.9 Angstrom resolution. The asymmetric unit consists of six noncrystallographic symmetry-related molecules that were exploited to improve initial multiple isomorphous replacement phasing, and subsequent structure refinement. The enzyme contains one disulfide bridge and is glycosylated at Asp164 by a single N-acetyl glucosamine residue. The protein has the expected fold for a glycoside hydrolase clan-C family 12 enzyme. It contains two beta -sheets, of six and nine strands, packed on top of one another, and one alpha -helix. The concave surface of the nine-stranded beta -sheet forms a large substrate-binding groove in which the active-site residues are located. In the active site, we find a carboxylic acid trio, similar to that of glycoside hydrolase families 7 and 16. The strictly conserved Asp99 hydrogen bonds to the nucleophile, the invariant Glu116. The binding crevice is lined with both aromatic and polar amino acid side-chains which may play a role in substrate binding. The structure of the fungal family 12 enzyme presented here allows a complete structural characterization of the glycoside hydrolase-C clan. (C) 2001 Academic Press.