Computational Design of Catalytic Dyads and Oxyanion Holes for Ester Hydrolysis

Abstract

Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion-holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by sidechains or bridging water molecules and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (kcat/KM) of 400 M−1s−1 for the cleavage of a p-nitrophenyl ester. Kinetic experiments indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.

Installing hydrolytic activity into a completely de novo protein framework

“…43,44 We envisage improving the efficiency and selectivity of our designs in the future through directed evolution of residues close to the active site, 12 and/or via the precise installation of a surrogate for the oxyanion hole to stabilize the tetrahedral intermediates. 19 In conclusion, this is the first report of catalytically active Cys-His-Glu triads being installed into a completely de novo protein framework. In total, seven such triads have been engineered into a heptameric -helical barrel, resulting in hydrolytic activities at least equal to those achieved in previous enzyme redesigns using natural scaffolds.…”

“…This "ping-pong" mechanism is well established for -chymotrypsin, which displays similar profiles with pNP esters and proceeds via an ester intermediate; 42 and it is similar to that proposed for previously designed hydrolase catalysts. 19 Rapid and stoichiometric acylation within the heptameric barrel leads to seven acylated Cys side chains, followed by rate-limiting hydrolysis of the thioester intermediate. In support of this mechanism, we could not find any evidence for acylation at His.…”

“…Whether the latter could adopt, or sample, different rotamers in solution was not clear from the X-ray crystal structure. 19 That 21 mutations, which corresponds to more than one third of the hydrophobic Ile or Leu residues, can be introduced into the lumen of CCHept is remarkable. These data highlight the hyperstability and mutability of these completely de novo -helical barrels.…”

“…For direct comparison to previous hydrolase designs, [17][18][19] we used a colorimetric p-nitrophenyl acetate (pNPA)-based assay to test for activity in the CC-Hept series. The single-mutant peptide assemblies, and hexameric CC-Hept-H-C-I showed no or little hydrolysis of pNPA above baseline at pH 7.0 (Fig.…”

“…[12][13][14][15][16] Computational approaches have also been used to design esterases, again with moderate activities being achieved through the placement of a histidine (His) residue into the active site of thioredoxin, 17 the C-terminal domain of calmodulin 18 and cysteine-His (Cys-His) dyads installed into existing / protein folds. 19 By contrast, much less has been demonstrated for the design of completely de novo catalysts; that is, where both the protein scaffold and the catalytic activity are built from scratch. [20][21][22] Although more challenging, this would carry advantages over redesign approaches: firstly, the designer could select and control all, or at least many of the residues, and so engineer the complete construct predictably; secondly, the resulting proteins could be made to function under conditions away from those required by natural proteins; and finally, success in this area would provide the acid test of our understanding of enzyme structure and function.…”