The road to fully programmable protein catalysis.

The ability to design efficient enzymes from scratch would have a profound effect on chemistry, biotechnology and medicine. Rapid progress in protein engineering over the past decade makes us optimistic that this ambition is within reach. The development of artificial enzymes containing metal cofactors and noncanonical organocatalytic groups shows how protein structure can be optimized to harness the reactivity of nonproteinogenic elements. In parallel, computational methods have been used to design protein catalysts for diverse reactions on the basis of fundamental principles of transition state stabilization. Although the activities of designed catalysts have been quite low, extensive laboratory evolution has been used to generate efficient enzymes. Structural analysis of these systems has revealed the high degree of precision that will be needed to design catalysts with greater activity. To this end, emerging protein design methods, including deep learning, hold particular promise for improving model accuracy. Here we take stock of key developments in the field and highlight new opportunities for innovation that should allow us to transition beyond the current state of the art and enable the robust design of biocatalysts to address societal needs.

Biosynthetic Functionalization of Nonribosomal Peptides.

Nonribosomal peptides (NRPs) are a therapeutically important class of secondary metabolites that are produced by modular synthetases in assembly-line fashion. We previously showed that a single Trp-to-Ser mutation in the initial Phe-loading adenylation domain of tyrocidine synthetase completely switches the specificity toward clickable analogues. Here we report that this minimally invasive strategy enables efficient functionalization of the bioactive NRP on the pathway level. In a reconstituted tyrocidine synthetase, the W227S point mutation permitted selective incorporation of Phe analogues with alkyne, halogen, and benzoyl substituents by the initiation module. The respective W2742S mutation in module 4 similarly permits efficient incorporation of these functionalized substrate analogues at position 4, expanding this strategy to elongation modules. Efficient incorporation of an alkyne handle at position 1 or 4 of tyrocidine A allowed site-selective one-step fluorescent labeling of the corresponding tyrocidine analogues by Cu(I)-catalyzed alkyne-azide cycloaddition. By combining synthetic biology with bioorthogonal chemistry, this approach holds great potential for NRP isolation and molecular target elucidation as well as combinatorial optimization of NRP therapeutics.

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 >104 turnovers per active site, exerts absolute control over reaction pathway and product stereochemistry, and displays a catalytic proficiency (1/KTS = 2.9 × 1010 M-1) that exceeds all previously characterized Diels-Alderases. These properties capitalize on effective Lewis acid catalysis, a chemical strategy for accelerating Diels-Alder reactions common in the laboratory but so far unknown in nature. Extension of this approach to other metal ions and other de novo scaffolds may propel the design field in exciting new directions.

Site-Specific Polymer Conjugation Stabilizes Therapeutic Enzymes in the Gastrointestinal Tract.

The site-specific conjugation of polymers to multiple engineered cysteine residues of a prolyl endopeptidase leads to its stabilization in the gastrointestinal tract of rats, without compromising the activity relative to the native enzyme. The importance of polymer attachment sites is investigated, as well as the significance of polymer structure.