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.

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.

An efficient pyrrolysyl-tRNA synthetase for economical production of MeHis-containing enzymes.

Genetic code expansion has emerged as a powerful tool in enzyme design and engineering, providing new insights into sophisticated catalytic mechanisms and enabling the development of enzymes with new catalytic functions. In this regard, the non-canonical histidine analogue Nδ-methylhistidine (MeHis) has proven especially versatile due to its ability to serve as a metal coordinating ligand or a catalytic nucleophile with a similar mode of reactivity to small molecule catalysts such as 4-dimethylaminopyridine (DMAP). Here we report the development of a highly efficient aminoacyl tRNA synthetase (G1PylRSMIFAF) for encoding MeHis into proteins, by transplanting five known active site mutations from Methanomethylophilus alvus (MaPylRS) into the single domain PylRS from Methanogenic archaeon ISO4-G1. In contrast to the high concentrations of MeHis (5-10 mM) needed with the Ma system, G1PylRSMIFAF can operate efficiently using MeHis concentrations of ∼0.1 mM, allowing more economical production of a range of MeHis-containing enzymes in high titres. Interestingly G1PylRSMIFAF is also a 'polyspecific' aminoacyl tRNA synthetase (aaRS), enabling incorporation of five different non-canonical amino acids (ncAAs) including 3-pyridylalanine and 2-fluorophenylalanine. This study provides an important step towards scalable production of engineered enzymes that contain non-canonical amino acids such as MeHis as key catalytic elements.

A non-canonical nucleophile unlocks a new mechanistic pathway in a designed enzyme.

Directed evolution of computationally designed enzymes has provided new insights into the emergence of sophisticated catalytic sites in proteins. In this regard, we have recently shown that a histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita-Baylis-Hillman (MBH) reaction with unrivalled efficiency. Here, we show that replacing the catalytic histidine with a non-canonical Nδ-methylhistidine (MeHis23) nucleophile leads to a substantially altered evolutionary outcome in which the catalytic Arg124 has been abandoned. Instead, Glu26 has emerged, which mediates a rate-limiting proton transfer step to deliver an enzyme (BHMeHis1.8) that is more than an order of magnitude more active than our earlier MBHase. Interestingly, although MeHis23 to His substitution in BHMeHis1.8 reduces activity by 4-fold, the resulting His containing variant is still a potent MBH biocatalyst. However, analysis of the BHMeHis1.8 evolutionary trajectory reveals that the MeHis nucleophile was crucial in the early stages of engineering to unlock the new mechanistic pathway. This study demonstrates how even subtle perturbations to key catalytic elements of designed enzymes can lead to vastly different evolutionary outcomes, resulting in new mechanistic solutions to complex chemical transformations.