A few snippets:
The overall distribution of fitness effects (DFE) measured during several DMS experiments across different target enzymes is consistent with the observations inferred from enzyme evolution campaigns: ~60-70% of mutations are deleterious, 30-40% are neutral, and less than 5% of mutations confer improvements in function.
Good news for the ID position? Not necessarily:
However, these observations must be put into perspective: assuming a protein sequence encompassing hundreds of amino acids, even a very small percentage of all available substitutions could still yield a substantial diversity of beneficial mutations. For example, nearly 2% of all possible single point substitutions in an enzyme of 300 amino acids (5,700 variants) would still correspond to 114 available beneficial mutations. Indeed, in a DMS study with VIM-2 β-lactamase, more than a hundred beneficial and specificity-altering mutations were identified across 25 different positions [Fig. 1(A)]. 24. This represents a significant reservoir of accessible beneficial mutations within the local sequence space that can be harnessed.
Other snippets that some may find interesting:
Whilst the fraction of beneficial mutations is largely consistent regardless of the enzyme model, the distribution of beneficial mutations on the tertiary structures seem to vary considerably among enzymes. In some cases, beneficial mutations cluster around the active site, e.g., in the DMS study of VIM-2 β-lactamase, 23 out of 25 positions that contain at least one specificity altering mutation were located within 15 Å of the catalytic zinc ions [Fig. 1(B)].24
In contrast, the majority of beneficial and specificity-determining mutations occurring in amiE appear located far from the active site: most of the 395 mutations that specifically enhanced growth on isobutyramide were 9-21 Å away from the active site [Fig. 1(B)].21 Similarly, 53/106 (50%) of mutations in TEM-1 β-lactamase that were found to increase fitness towards cefotaxime are localized on the enzyme surface and far from the active site.20
Overall, the wide distribution of activity-enhancing mutations on protein structures is likely a reflection of the existence of multiple solutions for improving interactions between an enzyme’s active site and its substrate. Mutations in the active site may generate critical residues that interact with the substrate and stabilize the TS, however, second-shell mutations may also help to fine-tune the key residues in the active site and/or binding pocket to be more complementary to the target substrate. In addition, surface mutations may function by altering conformational dynamics to more catalytically active conformations.
An intriguing question underlying the distribution of beneficial mutations is the extent to which epistasis influences the effects of mutations.35,36 For example, the effect of a mutation can switch from beneficial to deleterious (or the reciprocal), depending on the presence or absence of other mutations. Thus, as epistasis alters the nature of mutations, it restricts the accessibility of available substitutions and hence impacts evolutionary outcomes. On the scale of an entire protein sequence, epistasis appears relatively scarce, e.g., DMS studies conducted on the RNA recognition motif of poly(A)-binding protein and the IgG-binding domain of protein G found that pairwise epistasis occurred in only 4-5% of double mutants.37,38 By contrast, among activity enhancing mutations, epistasis appears highly prevalent and can drastically alter the effect of mutations.39-43 A systematic analysis of nine examples of enzyme evolution revealed that 82% of functional mutations exhibit epistasis, with nearly half appearing either neutral or deleterious in the wild-type background, only to become beneficial following the fixation of other mutations along the trajectory. 27
Perspectives and Future Directions
A growing number of studies have reported the evolution of enzymes performed in the laboratory or in nature. Here, we have highlighted over 30 examples that provide detailed molecular insights into the mechanisms of enzyme evolution (Table 1). Needless to say, each of these cases present unique and distinct genetic and molecular solutions that result from the incredible diversity of enzyme attributes, e.g., scaffold and active site architecture, type and level of native activity, and/or the acceptance of latent promiscuous substrates. Nonetheless, we observe several prominent mechanisms that are common amongst these examples of enzyme evolution. Importantly, we found that most of the examples herein do not rely on a single mechanism; rather, multiple strategies seem to be required to generate an efficient enzyme. In some cases, a defined sequence of events along a trajectory appears to be essential: initial mutations occur in the active site to generate a new interaction with the substrate, while later in the evolutionary process, distal mutations accumulate in the second-shell to tinker the position of the initial key residue in order to fine-tune its interaction with the novel substrate.25-27,33,112
[DMS=deep mutational scanning]
This same result is also reflected in many of the papers from the Thornton lab where they show, using ancestor reconstruction, that historical neutral mutations opened new pathways to subsequent adaptive mutations in both enzymes and various forms of structural and scaffolding proteins.