Insight into the significant roles of the Trp372 and flexible loop in directing the catalytic direction and substrate specificity in AGE superfamily enzymes

Abstract

The flexible loops or domains have been widely regarded to be involved in the catalytic modulation, substrate recognition, or thermal stability of enzymes. However, in AGE superfamily, the relationship of flexible loop and the tryptophan residue with catalytic direction and substrate specificity was still a mystery. In order to uncover this, the specific structures of cellobiose 2-epimerase (CE)-flexible loop and Trp372, were grafted into the mannose isomerase (MI) to generate a new recombinant protein named CMW. As a result, CMW showed no isomerization activity to monosaccharide-mannose but displayed epimerization and isomerization activity toward disaccharide, indicating that the CMW got close to CE but stepped far away from MI. These results demonstrated that the flexible loop and Trp372 were two key factors in determining the catalytic direction and substrate specificity. Combining the computational calculation and the structural analysis, it was speculated that: the (α/α)6-barrel of AGE superfamily only participated in the proton transfer process but didn’t guide the catalytic direction; in contrast, the epimerization activity was proposed to be determined by the tryptophan and the flexible loop and the isomerization was highly related to the residue composition and the shape change in the flexible loop. In addition, the substrate specificity for monosaccharide or disaccharide were suggested to be determined by the CsCE_Trp308 and Trp372.