10 rounds of DE for activity recovery were performed by error-prone PCR and activity screening of 350 enzyme variants by fluorescence assay in lysate. In vitro kinetics of purified variants indicated that DE recovered proteolytic activity by an improvement in both kobs1 (2x103-fold) and kobs2 (3x103-fold). Kinetic activity was recorded on purified enzyme in the presence of 1 µM substrate (pH 8, 25°C). Shown are kcat/KM for TEVCys variants (white circles) and kobs1 and kobs2 for TEVSER variants (black and grey circles respectively). The red dashed arrow indicates the effect of the initially introduced handicap nucleophile mutation (C151S). The nucleophile mutation did not affect TEVSer folding: the melting temperature (TM) of purified TEVSer was only marginally reduced (from 47.8 to 47.4 °C) and quantification of soluble expression in E. coli only decreased 10%. However, activity recovery in the TEVSer→TEVSerX DE trajectory correlated with reduced solubility (Fig. 2b) and stability analysis (using FoldX, Fig. 2c) indicated increasing destabilization. (b) Soluble expression of evolved variants (black) and S151C revertants (white) (measured by densitometric analysis of gel bands with ImageJ). (c) Energy difference between the folded and unfolded states (ΔG) for all evolved variants (black) and S151C revertants (white). Values are calculated by FoldX and normalized to TEVCys, higher values indicate lower stability. TOP: Shown are three residues which mutated during the DE lineage. Indicated is the mutation enrichment of any other amino acid. New residue evolved in DE lineage shown in grey boxes. The H28L mutation, introduced in TEVSerIV, showed enrichment of alternative residues in the two subsequent rounds, but is increasingly selected for retention in later rounds. The back mutation of W130C in TEVSerV is also predicted by its purge from the library. Finally the N174K of TEVSerVIII shows enrichment (of both N174K and N174Y) before it is selected in the lineage, after which further mutations are no longer enriched. LEFT: (a) Structure of TEVCys (PDB 1LVM) showing enrichment of non-wt amino acids for each residue averaged over whole TEVSer→TEVSerX lineage. Thick, blue regions indicate positive enrichment of non-wt amino acids, thin red region indicate purging of non-wt amino acids. Also shown are catalytic triad (red sticks) and N- terminal product (black sticks). (b) Proportion of residues that show enrichment >2-fold for each shell (normalized to the average for each round). Reversion of mutant TEVSerX shows unprecedented nucleophile permissiveness of the evolved variant. Although we deliberately evolved TEVCys via a fitness valley, the nucleophile-permissive TEVSerX can also be accessed from TEVCys by nearly-neutral mutations (Fig 7, left). Indeed TEVSerX shows 92.4% identity to TEVCys, as opposed to ~15% of closest serine proteases. The >103-fold improvement to generate a nucleophile generalist with so few mutations explains how divergent evolution of core catalytic machinery can occur within evolutionarily superfamilies such as the PA clan. The neutrality and abundance of these compensatory mutations may allow them to accumulate by drift before any nucleophile switch occurs and so act as ‘enabling’ mutations. A nucleophile mutation would be predicted to be more difficult to reconcile with two different types of bonds (C-O vs S-O) being formed and cleaved, compared to mutations to accommodate promiscuous binding of multiple substrates. However, our data suggest that the differences of nucleophile reactivity and structure can be readily accommodated by TEV protease and apparently little trade-off is necessary between nucleophiles. Previous attempts to quantify evolvability have found that the proportion of beneficial mutations lies in the range of one in 10-2 – 10-7 (depending on the organism and specific evolutionary challenge; Fig. 8, right). Our characterization of the local protein fitness landscapes puts the proportion of