Design and Analysis of Enhanced Catalysis in Scaffolded Multienzyme Cascade Reactions

Abstract

New developments in nucleic acid nanotechnology and protein scaffold designs have enabled unparalleled control over the spatial organization of synthetic multienzyme cascade reactions. One of the goals of these new technologies is to create nanostructured enzyme cascade reactions that promote substrate channeling along the cascade and, in doing so, enhance cascade catalysis. The concept of substrate channeling has a long and rich history in biochemistry and has established methods of evaluation and quantification. In this Perspective, we review the most common of these methods and discuss them in the context of engineered multienzyme systems and natural bifunctional enzymes with known mechanisms of substrate channeling. In addition, we use experimental data and the results of simulations of coupled-enzyme reactions to develop a set of preliminary design rules for engineering multienzyme nanostructures. The design rules address the limitations on interenzyme distance and active site orientation in substrate channeling and suggest designs for promoting enhanced catalysis, specifically, that enzyme orientation should minimize interenzyme distance and that at distances greater than 1 nm between active sites, significant channeling occurs only if diffusion of the intermediate is bounded through interactions with the surface or scaffold between active sites. This field is rapidly developing and promises to create many more new and exciting technologies.

Magnetic metal–organic frameworks as scaffolds for spatial co-location and positional assembly of multi-enzyme systems enabling enhanced cascade biocatalysis

“…3 Enlightened by the natural cellular metabolism, a series of multienzyme conjugates were developed, and signicant enhancements in the cascade reaction kinetics and efficiency both in vivo and in vitro have been reported. [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] Despite the massive interests, the assembly of "in vivo" multienzyme architecture and its full understanding are always difficult due to the extreme complexity of the intracellular environment.…”

Oxidative bioelectrocatalysis: From natural metabolic pathways to synthetic metabolons and minimal enzyme cascades

“…Nguyen et al used the sucrose oxidation enzymes discussed above to utilize DNA as a structural scaffold for altering the separation between sequential enzymes [19]. Although these studies have focused on proximity, non-bioelectrocatalytic studies have shown that substrate channeling is important [32]. Therefore, the future of enzyme cascade work will need to focus on developing synthetic metabolons that both provide proximity between active sites, and also provide for substrate channeling.…”

Designer Microorganisms for Optimized Redox Cascade Reactions – Challenges and Future Perspectives

“…On the other hand, these structural elements are also heterologous to the host organisma nd can contribute to the metabolicb urden.P reliminary design rules to control the number and orientation of enzymes in as patially nanostructured scaffold systemw ere clustered by Lin et al in three aspects:( i) inter-enzyme distance,( ii)a ctive site orientation,a nd (iii)m ultienzyme architecture. [134] During the last years,v arious innovative examples to create biomolecular nanostructures have been reported.T hese structures can be formed by proteinprotein interactions [135] as wella sn ucleic acids (DNA, [136] RNA [137] )o rp olymers (e.g.,c ell mimicking polymersomes [138] )( Figure 14). Theg roup of Keasling exemplified this approach by constructingaheterologous mevalonate pathway which contains hydroxyl-methylglutaryl-CoA synthase (HMGS)a nd hydroxyl-methylglutaryl-CoA reductase (HMGR)f rom S. cerevisiae)t ogether with the endogeneousa cetoactetyl-CoA transferase (AtoB)i nE.…”