Module recombination and functional integration of oligosaccharide-producing multifunctional amylase

Abstract

The oligosaccharide-producing multifunctional amylase (OPMA-N) has both hydrolytic and transglycosyl activities. Our previous reports demonstrated that the function and catalytic versatility of OPMA-N is closely related to its oligomerization, and its oligomeric state is affected by several conserved residues, such as Trp358, and by the cooperation of its small, noncatalytic N-terminal module and the catalytic module. We have demonstrated that the residue Trp358 exposed on the surface of OPMA-N molecule and has an obvious impact on OPMA-N oligomerization mainly by the charge effects. In this study, we investigated the effects of module recombination on the functional integration of OPMA-N. A series of module recombinants of the N-terminal and catalytic modules revealed that the intramolecular, semi-intramolecular and semi-intermolecular interactions of the N-terminal module with two or more catalytic modules enhanced the substrate affinity of the catalytic modules and facilitated the transglycosyl activity and functional integration of the enzyme by mediating positive cooperativity between the catalytic modules. Free N-terminal module alone did not contribute to these effects. Based on the results of this study, we speculated that the substrate affinity, but not the maximal catalytic activity, was the primary driving force in the natural evolution of enzymes, and that molecules of natural or modular enzymes may have occupied some evolutionary spaces that could be expanded or exhumed through module recombination to increase their overall catalytic efficacies or eventual catalytic efficiencies. All of the results in this study could be applied to integration of enzyme function and for creation of novel enzymes. Additionally, our results may provide important insights into the evolution of enzymes or organisms.