title: Crystal complex structures reveal how substrate is bound in the -4 to the +2 binding sites of Humicola grisea Cel12A.
abstract: As part of an ongoing enzyme discovery program to investigate the properties and catalytic mechanism of glycoside hydrolase family 12 (GH 12) endoglucanases, a GH family that contains several cellulases that are of interest in industrial applications, we have solved four new crystal structures of wild-type Humicola grisea Cel12A in complexes formed by soaking with cellobiose, cellotetraose, cellopentaose, and a thio-linked cellotetraose derivative (G2SG2). These complex structures allow mapping of the non-covalent interactions between the enzyme and the glucosyl chain bound in subsites -4 to +2 of the enzyme, and shed light on the mechanism and function of GH 12 cellulases. The unhydrolysed cellopentaose and the G2SG2 cello-oligomers span the active site of the catalytically active H.grisea Cel12A enzyme, with the pyranoside bound in subsite -1 displaying a S31 skew boat conformation. After soaking in cellotetraose, the cello-oligomer that is found bound in site -4 to -1 contains a beta-1,3-linkage between the two cellobiose units in the oligomer, which is believed to have been formed by a transglycosylation reaction that has occurred during the ligand soak of the protein crystals. The close fit of this ligand and the binding sites occupied suggest a novel mixed beta-glucanase activity for this enzyme.
authors.affiliation: Department of Cell and Molecular Biology, Uppsala University, Biomedical Center, Box 596, SE-751 24 Uppsala, Sweden. mats@xray.bmc.uu.se
authors.forename: Mats
authors.initials: M
authors.lastname: Sandgren
authors.forename: Gunnar I
authors.initials: GI
authors.lastname: Berglund
authors.forename: Andrew
authors.initials: A
authors.lastname: Shaw
authors.forename: Jerry
authors.initials: J
authors.lastname: Ståhlberg
authors.forename: Lennart
authors.initials: L
authors.lastname: Kenne
authors.forename: Tom
authors.initials: T
authors.lastname: Desmet
authors.forename: Colin
authors.initials: C
authors.lastname: Mitchinson
mesh_terms: D001203:Ascomycota / Q000201:enzymology*; D001665:Binding Sites; D002384:Catalysis; D002475:Cellobiose / Q000378:metabolism*; D002480:Cellulase / Q000737:chemistry* / Q000378:metabolism*; D002482:Cellulose / Q000031:analogs & derivatives* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D009844:Oligosaccharides / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013780:Tetroses / Q000378:metabolism*
chemical_list: D009844:Oligosaccharides; D013780:Tetroses; D002475:Cellobiose; C019193:maltopentaose; C048468:cellotetraose; D002482:Cellulose; D002480:Cellulase