The properties of the linker in a mini-scaffoldin influence the catalytic efficiency of scaffoldin-mediated enzyme complexes.

Synthetic enzyme complexes have been successfully used to accelerate the reaction rate of cascade enzyme biocatalysis. Protein scaffold-mediated enzyme complexes are often constructed by assembling cascade enzymes on the artificial protein scaffoldin to form sophisticated biomimetic architectures and enhance the catalytic efficiency of biocatalytic processes. However, the effects of the linker in scaffoldin on the performance of the enzyme complexes have not been clarified. In this study, a scaffoldin-mediated two-enzyme complex containing triosephosphate isomerase (TIM) and fructose-1,6-bisphosphate aldolase/phosphatase (FBPA) was constructed, and the initial production rate of fructose 6-phosphate (F6P) was determined with different types of fine-tuning linkers. Enzyme complexes with linker length of 25 amino acids in scaffoldin exhibited the highest initial F6P production rate compared with linker length of 0, 10, or 57 amino acids in scaffoldin. This result indicated that an appropriate interdomain spacing between functional domains was required by multienzyme complexes to facilitate effective cascade catalysis. Then, the most popular flexible linker GGGGS (unit F) and rigid linker EAAAK (unit R) were introduced into this 25 amino acid linker to investigate the effect of linker flexibility on the initial reaction rate of the TIM-FBPA enzyme complex. The synthetic enzyme complex with the semirigid linker FRRRF in scaffoldin showed the highest initial F6P production rate of 10.16 μM/min, which indicates that the linker's amino acid composition in scaffoldin may lead to significant changes in the spatial architecture of the TIM-FBPA complex and consequently affect the initial reaction rate. Precise linker length and flexibility allow an appropriate interdomain conformation to enable efficient cascade reactions. Collectively, our results showed that fine-tuning the initial reaction rate of enzyme complexes is an integrated systematic engineering, including adjusting the multienzyme architecture, linker length, and linker flexibility, which provides rational guidance for designing effective multienzyme complexes in the future.