Abstract
Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other.
Highlights
Multiple enzymes involved in related cellular processes are co-localized or clustered to form large non-covalent assemblies, thereby increasing local concentrations of intermediates near active sites[1,2,3,4]
In the cascade reaction consisting of Formate dehydrogenase (FDH) and Mannitol dehydrogenase (MDH) (Fig. 1b), NADH is regenerated by FDH-mediated formate oxidation, thereby continuously fueling MDHmediated D-mannitol production
In the presence of excess substrates for both enzymes, the transfer rate of NADH from FDH to MDH is supposed to govern the overall efficiency of the cascade reaction
Summary
Multiple enzymes involved in related cellular processes are co-localized or clustered to form large non-covalent assemblies, thereby increasing local concentrations of intermediates near active sites[1,2,3,4]. By systematically varying the distance between enzymes, they showed the multienzyme reaction displayed the distance-dependent efficiency, and attained great improvements when the enzymes were closely spaced as little as 10 nm Another important question on multienzyme systems is whether active site orientation affects cascade reaction efficiency. In case of the multienzyme systems constructed using nucleic acids or protein scaffolds[7,11], the active site of each enzyme had random or unidirectional orientations, hindering investigation of catalytic benefits from active sites directed towards one another in multienzyme cascade reactions. We described catalytic benefits from the controlled orientation of active sites of enzymes interacting in a cascade reaction To this end, we used site-specific incorporation of a reactive non-natural amino acid (NNAA) into enzymes followed by bioorthogonal enzyme-to-enzyme conjugation. We hypothesized that the active sites directed towards each other (face-to-face orientation) facilitate the intermediate transfer between enzymes more efficiently than the active sites directed away from each other (back-to-back orientation) (Fig. 1a)
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