Enzyme recruitment and its role in metabolic expansion

Abstract

Although more than 10(9) years have passed since the existence of the last universal common ancestor, proteins have yet to reach the limits of divergence. As a result, metabolic complexity is ever expanding. Identifying and understanding the mechanisms that drive and limit the divergence of protein sequence space impact not only evolutionary biologists investigating molecular evolution but also synthetic biologists seeking to design useful catalysts and engineer novel metabolic pathways. Investigations over the past 50 years indicate that the recruitment of enzymes for new functions is a key event in the acquisition of new metabolic capacity. In this review, we outline the genetic mechanisms that enable recruitment and summarize the present state of knowledge regarding the functional characteristics of extant catalysts that facilitate recruitment. We also highlight recent examples of enzyme recruitment, both from the historical record provided by phylogenetics and from enzyme evolution experiments. We conclude with a look to the future, which promises fruitful consequences from the convergence of molecular evolutionary theory, laboratory-directed evolution, and synthetic biology.

Figure 1

Genetic mechanisms that facilitate enzyme recruitment. (A) Gain-of-function point mutations (red) that endow new activity to an extant gene. (B) Beneficial point mutations (red) that afford enzyme overproduction by inactivating a repressor protein (R, yellow) or by disrupting the binding site of the repressor in the promoter (P) region. (C) Gene duplication. (D) Horizontal gene transfer by virtue of a phage or an extrachromosomal plasmid.

Figure 2

Properties that facilitate enzyme recruitment. (A) Trade-off between stability, which affords mutational robustness, and flexibility, which affords functional plasticity. (B) Substrate ambiguity (left) allows a single enzyme to transform multiple structurally distinct compounds, and catalytic promiscuity (right) allows a single enzyme to catalyze multiple chemically distinct transformations. (C) Epistasis constrains the evolutionary trajectories of ancestral enzyme sequences (black arrows represent mutations) into functionally discrete pools (blue, orange, and violet). Epistasis prevents the interconversion of contemporary functions without retracing past trajectories (red sign). Figure adapted from ref (61).

Figure 3

(A) Reactions catalyzed by atrazine chlorohydrolase (AtzA) and melamine deaminase (TriA), two enzymes that have 98% identical sequences. TriA possesses low levels of chlorohydrolase activity (red), but AtzA cannot catalyze the deaminase reaction. (B) Pericyclic reactions catalyzed by the highly homologous enzymes isochorismate pyruvate lyase (PchB) and chorismate mutase (CM). PchB catalyzes the chorismate mutase reaction at a low level (red), but CM is incapable of performing the PchB transformation.

Figure 4

Structures of representative molecules for which in vivo synthetic pathways have been successfully designed using enzyme recruitment: (A) nonane, (B) phenol, (C) the fluorinated triketide 5-fluoro-6-(1-fluoro-2-hydroxybutyl)-3-methyldihydro-2H-pyran-2,4(3H)-dione, (D) 3-hydroxypropionate, (E) styrene, and (F) 1,4-butanediol.

Figure 5

(A) Pathway design using enzymes recruited as a result of de novo design (red) that arise from ancestral reconstruction (green) or that possess promiscuous activities (blue). These catalysts can be installed into preexisting metabolic pathways (gray). (B) Structures of two pharmaceutical agents, Zytiga (Abiraterone) and Gilenya (Fingolimod), that represent attractive targets for future metabolic pathway design efforts with their annual treatment cost indicated.