Engineering of supramolecular nanoreactors by assembly of multiple enzymes for ATP regeneration in vitro

Abstract

The assembly of multiple enzymes has received increasing attention due to the promising application in improving biocatalytic process. In this study, supramolecular nanoreactors were engineered by assembly of polyphosphate kinase (PPK) and bifunctional glutathione synthetase (GshF) for adenosine triphosphate (ATP) regeneration in vitro. Tetrameric PPK and dimeric GshF were first self-assembled via peptide-peptide interaction of fused leucine zipper domains to form multienzyme complexes (MECs) with plate-like morphology. Further, under the driving force of polymerization of PPK driven by substrate polyphosphate (polyP), MECs were assembled into multienzyme nanoreactors (MENRs) with branch-like morphology. MENRs were applied to a two-enzyme cascade reaction in vitro. The ATP regeneration efficiency of MENRs system was 57 % higher than that of unassembled enzymes mixture, and the thermal stability was improved by 46 %. When reused for ATP regeneration, the assemblies retained more than 70 % of the initial activity even after seven cycles. This is the first report in which an artificial complex was constructed by substrate-driven assembly of multiple enzymes for cascade reaction. The strategy demonstrated potential application of multienzyme assembly in synthetic biotechnology and nanotechnology.