Crystal Complex Structures Reveal How Substrate is Bound in the −4 to the +2 Binding Sites of Humicola grisea Cel12A

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 (G 2SG 2). 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 G 2SG 2 cello-oligomers span the active site of the catalytically active H. grisea Cel12A enzyme, with the pyranoside bound in subsite −1 displaying a S 3 1 skew boat conformation. After soaking in cellotetraose, the cello-oligomer that is found bound in site −4 to −1 contains a β-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 β-glucanase activity for this enzyme.