Making Enzymes Suitable for Organic Chemistry by Rational Protein Design

Abstract

This review outlines recent developments in protein engineering of stereo-and regioselective enzymes, which are of prime interest in organic and pharmaceutical chemistry as well as biotechnology. The widespread application of enzymes was hampered for decades due to limited enantio-, diastereo-and regioselectivity, which was the reason why most organic chemists were not interested in biocatalysis. This attitude began to change with the advent of semi-rational directed evolution methods based on focused saturation mutagenesis at sites lining the binding pocket. Screening constitutes the laborintensive step (bottleneck), which is the reason why various research groups are continuing to develop techniques for the generation of small and smart mutant libraries. Rational enzyme design, traditionally an alternative to directed evolution, provides small collections of mutants which require minimal screening. This approach first focused on thermostabilization, and did not enter the field of stereoselectivity until later. Computational guides such as the Rosetta algorithms, HotSpot Wizard metric, and machine learning (ML) contribute significantly to decision making. The newest advancements show that semi-rational directed evolution such as CAST/ISM and rational enzyme design no longer develop on separate tracks, instead, they have started to merge. Indeed, researchers utilizing the two approaches have learned from each other. Today, the toolbox of organic chemists includes enzymes, primarily because the possibility of controlling stereoselectivity by protein engineering has ensured reliability when facing synthetic challenges. This review was also written with the hope that undergraduate and graduate education will include enzymes more so than in the past.[a] M. Reetz Max-Planck-Institut fur Kohlenforschung Mülheim an der Ruhr (Germany)

Ultrahigh-Throughput Enzyme Engineering and Discovery in In Vitro Compartments

“…125 Another focused library also led to rapid evolutionary improvements of a phosphotriesterase. 119 Indeed, such "smart" libraries 355,356 are often used to increase the chance of success of directed evolution campaigns. This has helped, especially when only lowthroughput screens were available, but the library design also limits the outcomes eventually.…”

Evaluating Multienzyme Cascade Routes for Pharmaceutically Relevant Molecules

“…To overcome these limitations, biotechnological advances have been made to engineer and artificially evolve the recombinant WT enzymes toward the desired functions. [19,20] Here, the recombinant enzymes can be engineered to remove, insert, or mutate specific amino acids in the enzyme structure. Various reports have demonstrated drastic changes in the enzyme properties upon protein engineering, including a complete switch in stereoselectivity, [21,22] expanded substrate scope, [23][24][25][26] enhanced activity [27] and thermostability, [28] tolerance to a broad range of pH [29] and different co-solvents, [30,31] and high substrate loading.…”

“…Most wild‐type (WT) enzymes display limited activity and stereoselectivity toward non‐natural substrates. To overcome these limitations, biotechnological advances have been made to engineer and artificially evolve the recombinant WT enzymes toward the desired functions [19,20] . Here, the recombinant enzymes can be engineered to remove, insert, or mutate specific amino acids in the enzyme structure.…”

Directing Protein Design Choices by Per-Residue Energy Breakdown Analysis with an Interactive Web Application

“…It holds the potential to revolutionize a wide range of applications (Arnold, 2018), from developing enzymes for industrial processes (Chen and Arnold, 2020), antibodies and antivirals for medicine (Sevy and Meiler, 2014; Willis et al, 2015) to molecular switches and biosensors (Stein and Alexandrov, 2015; Quijano-Rubio et al, 2021). Protein design has been demonstrated to play a crucial role in many different areas of biotechnology (Castro et al, 2022; Habibi et al, 2022; Reetz, 2022).…”