title: The X-ray crystal structure of the Trichoderma reesei family 12 endoglucanase 3, Cel12A, at 1.9 A resolution.
abstract: We present the three-dimensional structure of Trichoderma reesei endoglucanase 3 (Cel12A), 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 A resolution. The asymmetric unit consists of six non-crystallographic 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.
authors.affiliation: Department of Cell and Molecular Biology, Uppsala University, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Sandgren
authors.forename: A
authors.initials: A
authors.lastname: Shaw
authors.forename: T H
authors.initials: TH
authors.lastname: Ropp
authors.forename: S
authors.initials: S
authors.lastname: Wu
authors.forename: R
authors.initials: R
authors.lastname: Bott
authors.forename: A D
authors.initials: AD
authors.lastname: Cameron
authors.forename: J
authors.initials: J
authors.lastname: Ståhlberg
authors.forename: C
authors.initials: C
authors.lastname: Mitchinson
authors.forename: T A
authors.initials: TA
authors.lastname: Jones
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D002480:Cellulase / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000378:metabolism; D006031:Glycosylation; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D002480:Cellulase