Design and evolution of an enzyme with a non-canonical organocatalytic mechanism.

The combination of computational design and laboratory evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions1-4. However, the limited functionality presented by the genetic code restricts the range of catalytic mechanisms that are accessible in designed active sites. Inspired by mechanistic strategies from small-molecule organocatalysis5, here we report the generation of a hydrolytic enzyme that uses Nδ-methylhistidine as a non-canonical catalytic nucleophile. Histidine methylation is essential for catalytic function because it prevents the formation of unreactive acyl-enzyme intermediates, which has been a long-standing challenge when using canonical nucleophiles in enzyme design6-10. Enzyme performance was optimized using directed evolution protocols adapted to an expanded genetic code, affording a biocatalyst capable of accelerating ester hydrolysis with greater than 9,000-fold increased efficiency over free Nδ-methylhistidine in solution. Crystallographic snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. Nδ-methylhistidine can be considered to be a genetically encodable surrogate of the widely employed nucleophilic catalyst dimethylaminopyridine11, and its use will create opportunities to design and engineer enzymes for a wealth of valuable chemical transformations.

Strategies for designing biocatalysts with new functions.

The engineering of natural enzymes has led to the availability of a broad range of biocatalysts that can be used for the sustainable manufacturing of a variety of chemicals and pharmaceuticals. However, for many important chemical transformations there are no known enzymes that can serve as starting templates for biocatalyst development. These limitations have fuelled efforts to build entirely new catalytic sites into proteins in order to generate enzymes with functions beyond those found in Nature. This bottom-up approach to enzyme development can also reveal new fundamental insights into the molecular origins of efficient protein catalysis. In this tutorial review, we will survey the different strategies that have been explored for designing new protein catalysts. These methods will be illustrated through key selected examples, which demonstrate how highly proficient and selective biocatalysts can be developed through experimental protein engineering and/or computational design. Given the rapid pace of development in the field, we are optimistic that designer enzymes will begin to play an increasingly prominent role as industrial biocatalysts in the coming years.

Biocatalysis: landmark discoveries and applications in chemical synthesis.

Biocatalysis has become an important tool in chemical synthesis, allowing access to complex molecules with high levels of activity and selectivity and with low environmental impact. Key discoveries in protein engineering, bioinformatics, recombinant technology and DNA sequencing have contributed towards the rapid acceleration of the field. This tutorial review explores enzyme engineering strategies and high-throughput screening approaches that have been applied for the discovery and development of enzymes for synthetic application. Landmark developments in the field are discussed and have been carefully selected to highlight the diverse synthetic applications of enzymes within the pharmaceutical, agricultural, food and chemical industries. The design and development of artificial biocatalytic cascades is also examined. This tutorial review will give readers an insight into the landmark discoveries and milestones that have helped shape and grow this branch of catalysis since the discovery of the first enzyme.

Multicomponent Synthesis of the SARS-CoV-2 Main Protease Inhibitor Nirmatrelvir.

In the wake of the Covid-19 pandemic, it has become clear that global access to efficacious antiviral drugs will be critical to combat future outbreaks of SARS-CoV-2 or related viruses. The orally available SARS-CoV-2 main protease inhibitor nirmatrelvir has proven an effective treatment option for Covid-19, especially in compromised patients. We report a new synthesis of nirmatrelvir featuring a highly enantioselective biocatalytic desymmetrization (>99% ee) and a highly diastereoselective multicomponent reaction (>25:1 dr) as the key steps. Our route avoids the use of transition metals and peptide coupling reagents, resulting in an overall highly efficient and atom-economic process.

An Engineered Cytidine Deaminase for Biocatalytic Production of a Key Intermediate of the Covid-19 Antiviral Molnupiravir.

The Covid-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue recently approved as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of an efficient biocatalyst for the production of N-hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >85 000 turnovers in less than 3 h, operates at 180 g/L substrate loading, and benefits from in situ crystallization of the N-hydroxy-cytidine product (85% yield), which can be converted to Molnupiravir by a selective 5'-acylation using Novozym 435.

Engineering mutually orthogonal PylRS/tRNA pairs for dual encoding of functional histidine analogues.

The availability of an expanded genetic code opens exciting new opportunities in enzyme design and engineering. In this regard histidine analogues have proven particularly versatile, serving as ligands to augment metalloenzyme function and as catalytic nucleophiles in designed enzymes. The ability to genetically encode multiple functional residues could greatly expand the range of chemistry accessible within enzyme active sites. Here, we develop mutually orthogonal translation components to selectively encode two structurally similar histidine analogues. Transplanting known mutations from a promiscuous Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRSIFGFF ) into a single domain PylRS from Methanomethylophilus alvus (MaPylRSIFGFF ) provided a variant with improved efficiency and specificity for 3-methyl-L-histidine (MeHis) incorporation. The MaPylRSIFGFF clone was further characterized using in vitro biochemical assays and x-ray crystallography. We subsequently engineered the orthogonal MmPylRS for activity and selectivity for 3-(3-pyridyl)-L-alanine (3-Pyr), which was used in combination with MaPylRSIFGFF to produce proteins containing both 3-Pyr and MeHis. Given the versatile roles played by histidine in enzyme mechanisms, we anticipate that the tools developed within this study will underpin the development of enzymes with new and enhanced functions.

Engineering an efficient and enantioselective enzyme for the Morita-Baylis-Hillman reaction.

The combination of computational design and directed evolution could offer a general strategy to create enzymes with new functions. So far, this approach has delivered enzymes for a handful of model reactions. Here we show that new catalytic mechanisms can be engineered into proteins to accelerate more challenging chemical transformations. Evolutionary optimization of a primitive design afforded an efficient and enantioselective enzyme (BH32.14) for the Morita-Baylis-Hillman (MBH) reaction. BH32.14 is suitable for preparative-scale transformations, accepts a broad range of aldehyde and enone coupling partners and is able to promote selective monofunctionalizations of dialdehydes. Crystallographic, biochemical and computational studies reveal that BH32.14 operates via a sophisticated catalytic mechanism comprising a His23 nucleophile paired with a judiciously positioned Arg124. This catalytic arginine shuttles between conformational states to stabilize multiple oxyanion intermediates and serves as a genetically encoded surrogate of privileged bidentate hydrogen-bonding catalysts (for example, thioureas). This study demonstrates that elaborate catalytic devices can be built from scratch to promote demanding multi-step processes not observed in nature.

Enzymes with noncanonical amino acids.

Enzyme design and engineering strategies rely almost exclusively on nature's alphabet of twenty canonical amino acids. Recent years have seen the emergence of powerful genetic code expansion methods that allow hundreds of structurally diverse amino acids to be installed into proteins in a site-selective manner. Here, we will highlight how the availability of an expanded alphabet of amino acids has opened new avenues in enzyme engineering research. Genetically encoded noncanonical amino acids have provided new tools to probe complex enzyme mechanisms, improve biocatalyst activity and stability, and most ambitiously to design enzymes with new catalytic mechanisms that would be difficult to access within the constraints of the genetic code. We anticipate that the studies highlighted in this article, coupled with the continuing advancements in genetic code expansion technology, will promote the widespread use of noncanonical amino acids in biocatalysis research in the coming years.