Enzyme Evolution


Life involves an enormous diversity of coupled chemical reactions, almost none of which would occur in a biologically relevant time scale without catalysis. Enzymes are life catalysts, capable of enhancing the rates of biochemical reactions by many orders of magnitude. Modern natural enzymes are the complex outcome of evolution operating over a vast expanse of time. Plausibly, the overall process started ∼4 billion years ago when polypeptides that possibly served as cofactors of ribozymes in the RNA world acquired the capability to catalyse simple reactions. Likely, primordial enzymes were generalists that could catalyse various reactions with moderate efficiency. Diversification, specialisation and optimisation occurred subsequently over evolutionary history, probably coupled to successive gene duplication events. Advances in protein engineering and laboratory evolution have allowed some of the main stages in this evolutionary narrative to be reproduced in the laboratory and have demonstrated the fundamental role of conformational diversity in enzyme evolution.

Key Concepts

  • Modern natural enzymes are the complex outcome of evolution operating over ∼4 billion years.
  • Most modern natural enzymes evolved from previously existing enzymes.
  • To avoid detrimental effects on organismal fitness, the emergence of new enzymes from old enzymes is likely coupled to gene duplication.
  • Directed evolution experiments allow the transformation of an old enzyme into a new enzyme to be reproduced in the laboratory.
  • The evolutionary transformation of an old enzyme into a new enzyme likely occurs trough multifunctional intermediates.
  • Unless we accept panspermia as an explanation for the origin of life on Earth, we must admit that, at some very early stage of life evolution on this planet, primordial enzymes emerged de novo in noncatalytic scaffolds.
  • Recent work has shown that single mutations can generate emerging enzyme functionalities in previously noncatalytic scaffolds.
  • Proteins in solution are best envisioned as ensembles of different conformations.
  • Experimental and computational studies support that conformational diversity underlies enzyme ‘evolvability’, that is, the capability to evolve towards new functionalities.
  • Our current understanding of enzyme evolution is not detailed enough to provide a reliable basis for rational enzyme design.

Keywords: Enzyme evolution; enzyme promiscuity; de novo enzymes; protein evolvability; gene duplication; laboratory directed evolution; conformational diversity; structural dynamics

Figure 1. General model of enzyme evolution mediated by gene duplication and conformational diversity. (a) The protein populates an ensemble of different conformations. The major conformer binds the native substrate (yellow) and is responsible for the original function. (b) A minor alternative conformer can bind a different substrate (pink) and is responsible for a low‐level promiscuous activity. Mutations that shift the conformational equilibria towards the alternative conformer will enhance the promiscuous activity but will impair the original activity, thus compromising organismal fitness. (c) Gene duplication avoids such detrimental effects on organismal fitness, as one copy of the gene maintains the old function while the other copy accepts mutations that shift the conformational equilibria and evolves towards increasing levels of the new function. James and Tawfik . Reproduced with permission from Elsevier.
Figure 2. Native versus promiscuous enzyme activities. A natural phosphotriesterase capable to efficiently degrade paraoxon (a) also displays a low‐level promiscuous arylesterase activity and is able to degrade, although rather inefficiently, 2‐naphthyl hexanoate (b).
Figure 3. Laboratory directed evolution of an arylesterase (new function) from a paraoxonase (old function) (Tokuriki et al., ). See Figure for the structures of the substrates involved. (a) Catalytic efficiencies for the two activities for purified enzyme variants over the several rounds of the laboratory evolution experiment. It is clear that the initial and final states of the experiment correspond to specialist enzymes. On the other hand, the enzymes at some intermediate states are multifunctional and show moderately efficient catalysis for both reactions. (b) Plot of paraoxonase activity versus arylesterase activity over the rounds of the laboratory evolution experiment. Trade‐off between the two activities is not observed in the first few rounds. Eventually, however, increases in arylesterase activity bring about a strong decrease in the native, paraoxonase activity. In a natural evolution scenario, gene duplication (Figure ) would be required to avoid detrimental effects on organismal fitness.
Figure 4. A common benchmark in the de novo enzyme design. (a) Kemp elimination of 5‐nitrobenzioxazole showing the catalytic base and a proposed transition state structure. Kemp elimination is a simple model of proton abstraction from carbon, a fundamental process in chemistry and biochemistry, and has been extensively used as benchmark in de novo enzyme design. (b) Tryptophan. (c) 5(6)‐Nitrobenzotriazole, a transition‐state analogue for Kemp elimination. (d) Indole, the tryptophan side chain. Risso et al. . Licensed under CC BY 4.0.
Figure 5. Structures of de novo Kemp eliminases generated using a single‐mutation, minimalist design. Since the Kemp substrate has a shape similar to that of tryptophan side chain (Figure ), replacing W229 with aspartate generates, both a suitable cavity and a catalytic base at its bottom. (a) and (b) show the structures of two catalytic W229D variants with a bound transition‐state analogue superimposed with the structures of their corresponding background scaffolds. Displacement of two alpha‐helices is required to allow transition state binding. The background scaffold in (b) is pre‐organised and displays the required displacement before the catalysis generating mutation has been introduced. This is more clearly seen in the comparison between the two background scaffolds shown in (c). (d) and (e) show blow‐ups of the de novo active site regions for the Kemp eliminases in (a) and (b). Risso et al. . Licensed under CC BY 4.0.


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Further Reading

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Sanchez‐Ruiz, Jose M(Oct 2019) Enzyme Evolution. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0028797]