The evolutionary advantage of an aromatic clamp in plant family 3 glycoside exo-hydrolases

Barley beta-d-glucan glucohydrolase is a glycoside hydrolase family 3 (GH3) enzyme critical for growth and development. Here the authors carryout mutagenesis, structural analyses and multi-scale molecular dynamics to examine the binding and conformational behaviour of several beta-d-glucosides during the substrate-product assisted catalysis that operates in GH3 hydrolases. In the barley beta-d-glucan glucohydrolase, a glycoside hydrolase family 3 (GH3) enzyme, the Trp286/Trp434 clamp ensures beta-d-glucosides binding, which is fundamental for substrate hydrolysis during plant growth and development. We employ mutagenesis, high-resolution X-ray crystallography, and multi-scale molecular modelling methods to examine the binding and conformational behaviour of isomeric beta-d-glucosides during substrate-product assisted processive catalysis that operates in GH3 hydrolases. Enzyme kinetics reveals that the W434H mutant retains broad specificity, while W434A behaves as a strict (1,3)-beta-d-glucosidase. Investigations of reactant movements on the nanoscale reveal that processivity is sensitive to mutation-specific alterations of the tryptophan clamp. While wild-type and W434H utilise a lateral cavity for glucose displacement and sliding of (1,3)-linked hydrolytic products through the catalytic site without dissociation, consistent with their high hydrolytic rates, W434A does not adopt processive catalysis. Phylogenomic analyses of GH3 hydrolases disclose the evolutionary advantage of the tryptophan clamp that confers broad specificity, high catalytic efficiency, and processivity.

Automated summary

In this study, the researchers investigate the binding and conformational behavior of beta-D-glucosides in the barley beta-D-glucan glucohydrolase. They find that the Trp286/Trp434 clamp plays a crucial role in substrate hydrolysis. Mutations in the clamp affect processive catalysis, and phylogenomic analysis reveals the evolutionary advantage of the tryptophan clamp.