Epistasis arises from shifting the rate-limiting step during enzyme evolution of a β-lactamase.

Epistasis, the non-additive effect of mutations, can provide combinatorial improvements to enzyme activity that substantially exceed the gains from individual mutations. Yet the molecular mechanisms of epistasis remain elusive, undermining our ability to predict pathogen evolution and engineer biocatalysts. Here we reveal how directed evolution of a β-lactamase yielded highly epistatic activity enhancements.

Electric Fields Are a Key Determinant of Carbapenemase Activity in Class A β-Lactamases.

Resistance to antibiotics is a public health crisis. Although carbapenems are less susceptible to resistance than other β-lactam antibiotics, β-lactamases mediating resistance against these drugs are spreading. Here, we dissect the contributions of electric fields to carbapenemase activity in class A β-lactamases.

Selection of a promiscuous minimalist cAMP phosphodiesterase from a library of de novo designed proteins.

The ability of unevolved amino acid sequences to become biological catalysts was key to the emergence of life on Earth. However, billions of years of evolution separate complex modern enzymes from their simpler early ancestors. To probe how unevolved sequences can develop new functions, we use ultrahigh-throughput droplet microfluidics to screen for phosphoesterase activity amidst a library of more than one million sequences based on a de novo designed 4-helix bundle.

An expandable, modular de novo protein platform for precision redox engineering.

The electron-conducting circuitry of life represents an as-yet untapped resource of exquisite, nanoscale biomolecular engineering. Here, we report the characterization and structure of a de novo diheme "maquette" protein, 4D2, which we subsequently use to create an expanded, modular platform for heme protein design. A well-folded monoheme variant was created by computational redesign, which was then utilized for the experimental validation of continuum electrostatic redox potential calculations.

Rigidifying a Enzyme Increases Activity and Induces a Negative Activation Heat Capacity.

Conformational sampling profoundly impacts the overall activity and temperature dependence of enzymes. Peroxidases have emerged as versatile platforms for high-value biocatalysis owing to their broad palette of potential biotransformations. Here, we explore the role of conformational sampling in mediating activity in the peroxidase C45.

Evolution of dynamical networks enhances catalysis in a designer enzyme.

Activation heat capacity is emerging as a crucial factor in enzyme thermoadaptation, as shown by the non-Arrhenius behaviour of many natural enzymes. However, its physical origin and relationship to the evolution of catalytic activity remain uncertain. Here we show that directed evolution of a computationally designed Kemp eliminase reshapes protein dynamics, which gives rise to an activation heat capacity absent in the original design.

Efficient Lewis acid catalysis of an abiological reaction in a de novo protein scaffold.

New enzyme catalysts are usually engineered by repurposing the active sites of natural proteins. Here we show that design and directed evolution can be used to transform a non-natural, functionally naive zinc-binding protein into a highly active catalyst for an abiological hetero-Diels-Alder reaction. The artificial metalloenzyme achieves >10 turnovers per active site, exerts absolute control over reaction pathway and product stereochemistry, and displays a catalytic proficiency (1/K = 2.

Designing better enzymes: Insights from directed evolution.

De novo enzymes can be created by computational design and directed evolution. Here, we review recent insights into the origins of catalytic power in evolved designer enzymes to pinpoint opportunities for next-generation designs: Evolution precisely organizes active sites, introduces catalytic H-bonding networks, invokes electrostatic catalysis, and creates dynamical networks embedding the active site in a reactive protein scaffold. Such insights foster our fundamental knowledge of enzyme catalysis and fuel the future design of tailor-made enzymes.

How directed evolution reshapes the energy landscape in an enzyme to boost catalysis.

The advent of biocatalysts designed computationally and optimized by laboratory evolution provides an opportunity to explore molecular strategies for augmenting catalytic function. Applying a suite of nuclear magnetic resonance, crystallography, and stopped-flow techniques to an enzyme designed for an elementary proton transfer reaction, we show how directed evolution gradually altered the conformational ensemble of the protein scaffold to populate a narrow, highly active conformational ensemble and accelerate this transformation by nearly nine orders of magnitude. Mutations acquired during optimization enabled global conformational changes, including high-energy backbone rearrangements, that cooperatively organized the catalytic base and oxyanion stabilizer, thus perfecting transition-state stabilization.

Emergence of a Negative Activation Heat Capacity during Evolution of a Designed Enzyme.

Temperature influences the reaction kinetics and evolvability of all enzymes. To understand how evolution shapes the thermodynamic drivers of catalysis, we optimized the modest activity of a computationally designed enzyme for an elementary proton-transfer reaction by nearly 4 orders of magnitude over 9 rounds of mutagenesis and screening. As theorized for primordial enzymes, the catalytic effects of the original design were almost entirely enthalpic in origin, as were the rate enhancements achieved by laboratory evolution.

Speeding up enzyme discovery and engineering with ultrahigh-throughput methods.

Exploring the sequence space of enzyme catalysts is ultimately a numbers game. Ultrahigh-throughput screening methods for rapid analysis of millions of variants are therefore increasingly important for investigating sequence-function relationships, searching large metagenomic libraries for interesting activities, and accelerating enzyme evolution in the laboratory. Recent applications of such technologies are reviewed here, with a particular focus on the practical benefits of droplet-based microfluidics for the directed evolution of natural and artificial enzymes.