Artificial Multienzyme Supramolecular Device: Highly Ordered Self‐Assembly of Oligomeric Enzymes In Vitro and In Vivo

Abstract

A strategy for scaffold-free self-assembly of multiple oligomeric enzymes was developed by exploiting enzyme oligomerization and protein-protein interaction properties, and was tested both in vitro and in vivo. Octameric leucine dehydrogenase and dimeric formate dehydrogenase were fused to a PDZ (PSD95/Dlg1/zo-1) domain and its ligand, respectively. The fusion proteins self-assembled into extended supramolecular interaction networks. Scanning-electron and atomic-force microscopy showed that the assemblies assumed two-dimensional layer-like structures. A fluorescence complementation assay indicated that the assemblies were localized to the poles of cells. Moreover, both in vitro and in vivo assemblies showed higher NAD(H) recycling efficiency and structural stability than did unassembled structures when applied to a coenzyme recycling system. This work provides a novel method for developing artificial multienzyme supramolecular devices and for compartmentalizing metabolic enzyme cascades in living cells.

Engineering bi‐functional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration

“…Gao et al. fused LeuDH and FDH with PDZ (PSD95/Dlg1/zo‐1) domain and corresponding ligand (PDZlig) from metazoan cells respectively for in vitro and vivo self‐assembly of multienzyme supramolecular. Both in vitro and vivo assemblies exhibited higher cofactor regeneration efficiency and structural stability than free enzyme mixture.…”

Construction and Characterization of a Novel Glucose Dehydrogenase-Leucine Dehydrogenase Bifunctional Enzyme for the Biosynthesis of L-Tert-Leucine

“…Firstly, by analyzing the PDB structure, we determined the sequence fusion sequence from GDH to LeuDH, so that the integrity of the oligomerization interface of each oligomerase could be maintained. Then, in order to prevent the two enzymes from interacting with ea ch other during the folding process, it is essential to add a peptide link to mediate [18,27].…”

“…Inspired by sophisticated protein complexes in nature [15,16], arti cial multi-enzyme systems have been reported to improve structural stability, promote the cofactor regeneration e ciency and reduce the cost of free enzyme system [17,18]. LeuDH and FDH with PDZ (PSD95/Dlg1/Zo-1) domain and the corresponding ligand of the metazoan (PDZlig) were fused and assembled as multiple supramolecules to improve the NAD(H) recycling e ciency and structural stability [19].…”

Computation‐Guided Design of a Stimulus‐Responsive Multienzyme Supramolecular Assembly

“…Enzyme clustering can lead to higher metabolic flux as a result of substrate channeling, sequestration of toxic intermediates, and decreased competing side reactions . Based on this natural blueprint, the design of artificial multienzyme metabolons has been attempted both in vitro and in vivo by using a host of colocalization strategies . Although these strategies were successful in improving the efficiency of synthetic metabolic pathways to various extents, no temporal control, which is characteristic of natural metabolic assemblies, such as the purinosome, on the formation of the multienzyme complexes has been designed.…”

“…The increase due to colocalization was insensitive to changes in the enzyme stoichiometry used for assembly, thus indicating that assembly formation was robust (Figure S12). To evaluate whether the colocalization of enzymes affords other functional benefits such as enhanced thermotolerance, an important parameter for the utility of synthetic metabolic pathways, we evaluated the ability of assembled enzymes and unassembled controls to tolerate a heat shock. GLY production yield at room temperature was measured after 30 min of exposure to a range of temperatures, and we found that the three‐component assembly maintains activity to a greater extent than unassembled enzymes do (Figure C).…”