Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59

Abstract

Computational design is a test of our understanding of enzyme catalysis and a means of engineering novel, tailor-made enzymes. While the de novo computational design of catalytically efficient enzymes remains a challenge, designed enzymes may comprise unique starting points for further optimization by directed evolution. Directed evolution of two computationally designed Kemp eliminases, KE07 and KE70, led to low to moderately efficient enzymes (k cat ∕K m values of ≤5 × 10 4 M −1 s −1 ). Here we describe the optimization of a third design, KE59. Although KE59 was the most catalytically efficient Kemp eliminase from this design series (by k cat ∕K m , and by catalyzing the elimination of nonactivated benzisoxazoles), its impaired stability prevented its evolutionary optimization. To boost KE59's evolvability, stabilizing consensus mutations were included in the libraries throughout the directed evolution process. The libraries were also screened with less activated substrates. Sixteen rounds of mutation and selection led to >2,000-fold increase in catalytic efficiency, mainly via higher k cat values. The best KE59 variants exhibited k cat ∕K m values up to 0.6 × 10 6 M −1 s −1 , and k cat ∕k uncat values of ≤10 7 almost regardless of substrate reactivity. Biochemical, structural, and molecular dynamics (MD) simulation studies provided insights regarding the optimization of KE59. Overall, the directed evolution of three different designed Kemp eliminases, KE07, KE70, and KE59, demonstrates that computational designs are highly evolvable and can be optimized to high catalytic efficiencies.computational protein design | enzyme mimic T he endeavor of making enzyme-like catalysts spans several decades (1) with the Kemp elimination being a thoroughly explored model (2-7). In this activated model system, basecatalyzed proton elimination from carbon is concerted with the cleavage of nitrogen-oxygen bond, thus leading to the cyanophenol product (Fig. 1A). Activation of a carboxylate base catalyst by desolvation is efficiently mimicked by aprotic dipolar solvents such as acetonitrile and by various enzyme mimics. Effective alignment of the substrate and charge-dispersing interactions that stabilize the negatively charged transition state (TS) have also been achieved by various enzyme mimics that catalyze the Kemp elimination (8, 9). However, generation of a (wo)manmade active site that exhibits all these features and performs as well as natural enzymes, remains a challenge.Computational methods for predicting structure from sequence at atomic accuracy provide a new approach to enzyme engineering (10-12). The design involves two steps: (i) designing an active-site configuration that may confer efficient catalysis, (ii) computing a sequence that confers the desired configuration. Both steps are currently far from optimal, as the catalytic efficiency of designed enzymes falls far behind that of natural enzymes. Further, as with other enzyme mimics, computational designs tackle activated model systems and fail to catalyze cha...

Engineering a model protein cavity to catalyze the Kemp elimination

“…As impressive as recent design efforts have been, the resulting enzymes have retained much of the complexity of the templates on which they were built, and have resisted full analysis of how structure encodes function (35). By comparison, in these cavities, the origins of catalysis seem simpler and the specific groups and geometries that contribute to it are readily grasped.…”

Computational Enzyme Design

“…The k cat /K M value of KE07 was improved 200-fold, [185] that of KE70 over 400-fold, [186] and in the case of KE59 the k cat /K M value was increased over 2000-fold. [187] The studies demonstrate how computational protein design can be used to generate enzymes with modest activities that can then be further optimized through directed evolution approaches.…”

Computational Optimization of Electric Fields for Improving Catalysis of a Designed Kemp Eliminase

“…kcat/KM values of 2.0x10 3 , 5.7x10 4 , 6.0x10 5 , and 2.3x10 6 M -1 s -1 , respectively, which has led to much optimism for achieving efficiencies that rival natural enzymes.…”

“…De novo designed enzymes via computer modeling of active sites accommodated within a variety of protein scaffolds [1][2] , have been reported for reactions such Kemp elimination [3][4][5][6] , retroaldol condensation 7 and Diels-Alders chemistry 8 . However, most computational designs to date have exhibited very little catalytic competence compared to native enzymes or enzymes subsequently improved through laboratory directed evolution (LDE).…”

“…A case in point is the Kemp elimination reaction, a one-step proton transfer reaction from the 5-nitrobenzisoxazole substrate by a catalytic base, leading to breaking of the 5-membered ring and forming the final product, alpha-cyanophenol ( Figure 1a). Various Kemp eliminases that were computationally designed catalyze this reaction with efficiencies as measured by kcat/KM of 12, 126, 160, and 425 M -1 s -1 , for KE07 5 , KE70 6 , KE59 4 , and HG3 9 , respectively. However substantial gain in biocatalytic activity is achieved when these minimal designs are subjected to laboratory directed evolution LDE; after undergoing LDE, the Kemp eliminases KE07 5 , KE70 6 , KE59 4 , and HG3 10 yielded…”