Crystal structure of a fungal glycoside hydrolase family 3 β-glucosidase, ReCel3A from the moderately thermophilic fungus Rasamsonia emersonii

A glycoside hydrolase (GH) family 3 β-glucosidase from the moderately thermophilic Rasamsonia emersonii, TeCel3A, was heterologously expressed in Hypocrea jecorina. The expressed protein was purified, crystallized and the structure of the enzyme was solved by x-ray crystallography to 2.2 Å resolution. The structure was solved by molecular replacement. This is to our knowledge the first structure of a three-domain GH family 3 β-glucosidase that contains a novel feature in the form of a very extended loop insertion in the middle of the third, C-terminal domain. This loop folds back on top of and follows the surface of the N-terminal domain, which contains the substrate-binding pocket of the enzyme. A second ligand bound structure of the enzyme with β-D-glucose bound in the active site of the enzyme, was also solved to 2.6 Å resolution. There are a total four non-crystallographic symmetry (NCS) related molecules in the ReCel3A crystal structure. These four NCS molecules form two apparent dimers in the structure. This finding is in consistence with previously published result that ReCel3a often appear as dimers in solution¹. The TeCel3A structure is a three domain structure that consists of an N-terminal (α/β)₈ TIM barrel domain, providing the platform for most of the active site, as well as providing the the catalytic nucleophile, followed by an (α/β)₆ sandwich domain, which provides the acid/base amino acid residue of the active site, and finally the structure has a third C-terminal domain which has a fibronectin type III fold, with of yet unknown function.