Robust design and optimization of retroaldol enzymes

Abstract

Enzyme catalysts of a retro-aldol reaction have been generated by computational design using a motif that combines a lysine in a non-polar environment with water-mediated stabilization of the carbinolamine hydroxyl and β-hydroxyl groups. Here we show that the design process is robust and repeatable, with 33 new active designs constructed on 13 different protein scaffold backbones. The initial activities are not high but are increased through site-directed mutagenesis and laboratory evolution. Mutational data highlight areas for improvement in design. Different designed catalysts give different borohydride-reduced reaction intermediates, suggesting a distribution of properties of the designed enzymes that may be further explored and exploited.

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

“…459 Notable examples include a metalloenzyme for organophosphate hydrolysis, 460,461 aldolase 462,463 and others. 464–468 Theozymes 469–472 (theoretical catalysts, constructed by computing the optimal geometry for transition state stabilization by model functional) groups represent another approach.…”

Exploration of Alternate Catalytic Mechanisms and Optimization Strategies for Retroaldolase Design

“…Multistep retroaldol reactions, which are subject to amine catalysis, were among the first transformations tackled by computational design [2,7]. Catalysis is initiated by attack of a reactive lysine on the carbonyl group of the β-hydroxy-ketone substrate to form a tetrahedral carbinolamine intermediate that subsequently breaks down to give a protonated Schiff base.…”

Computational strategies for the design of new enzymatic functions

“…In an attempt to improve the efficiency of the new designed enzymes, Hilvert, Baker and co-workers optimized the protein via directed evolution, 114,115 from the previously computationally retro-aldolases that were designed from scratch. 19 The specific activity was boosted more than 4,400-fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes.…”