The incorporation of a synthetic, catalytically competent metallocofactor into a protein scaffold to generate an artificial metalloenzyme (ArM) has been explored since the late 1970's. Progress in the ensuing years was limited by the tools available for both organometallic synthesis and protein engineering. Advances in both of these areas, combined with increased appreciation of the potential benefits of combining attractive features of both homogeneous catalysis and enzymatic catalysis, led to a resurgence of interest in ArMs starting in the early 2000's. Perhaps the most intriguing of potential ArM properties is their ability to endow homogeneous catalysts with a genetic memory. Indeed, incorporating a homogeneous catalyst into a genetically encoded scaffold offers the opportunity to improve ArM performance by directed evolution. This capability could, in turn, lead to improvements in ArM efficiency similar to those obtained for natural enzymes, providing systems suitable for practical applications and greater insight into the role of second coordination sphere interactions in organometallic catalysis. Since its renaissance in the early 2000's, different aspects of artificial metalloenzymes have been extensively reviewed and highlighted. Our intent is to provide a comprehensive overview of all work in the field up to December 2016, organized according to reaction class. Because of the wide range of non-natural reactions catalyzed by ArMs, this was done using a functional-group transformation classification. The review begins with a summary of the proteins and the anchoring strategies used to date for the creation of ArMs, followed by a historical perspective. Then follows a summary of the reactions catalyzed by ArMs and a concluding critical outlook. This analysis allows for comparison of similar reactions catalyzed by ArMs constructed using different metallocofactor anchoring strategies, cofactors, protein scaffolds, and mutagenesis strategies. These data will be used to construct a searchable Web site on ArMs that will be updated regularly by the authors.

Artificial metalloenzymes (ArMs) catalyzing new-to-nature reactions could play an important role in transitioning toward a sustainable economy. While ArMs have been created for various transformations, attempts at their genetic optimization have been case specific and resulted mostly in modest improvements. To realize their full potential, methods to rapidly discover active ArM variants for ideally any reaction of interest are required. Here, we introduce a reaction-independent, automation-compatible platform, which relies on periplasmic compartmentalization in Escherichia coli to rapidly and reliably engineer ArMs based on the biotin-streptavidin technology. We systematically assess 400 ArM mutants for five bioorthogonal transformations involving different metals, reaction mechanisms, and reactants, which include novel ArMs for gold-catalyzed hydroamination and hydroarylation. Activity enhancements up to 15-fold highlight the potential of the systematic approach. Furthermore, we suggest smart screening strategies and build machine learning models that accurately predict ArM activity from sequence, which has crucial implications for future ArM development.

Artifical metalloenzymes combine the reactivity of small molecule catalysts with the selectivity of enzymes, and new methods are required to tune the catalytic properties of these systems for an application of interest. Structure-based computational design could help to identify amino acid mutations leading to improved catalytic activity and enantioselectivity. Here we describe the application of Rosetta Design for the genetic optimization of an artificial transfer hydrogenase (ATHase hereafter), [(η(5)-Cp*)Ir(pico)Cl] ⊂ WT hCA II (Cp* = Me5C5(-)), for the asymmetric reduction of a cyclic imine, the precursor of salsolsidine. Based on a crystal structure of the ATHase, computational design afforded four hCAII variants with protein backbone-stabilizing and hydrophobic cofactor-embedding mutations. In dansylamide-competition assays, these designs showed 46-64-fold improved affinity for the iridium pianostool complex [(η(5)-Cp*)Ir(pico)Cl]. Gratifyingly, the new designs yielded a significant improvement in both activity and enantioselectivity (from 70% ee (WT hCA II) to up to 92% ee and a 4-fold increase in total turnover number) for the production of (S)-salsolidine. Introducing additional hydrophobicity in the Cp*-moiety of the Ir-catalyst provided by adding a propyl substituent on the Cp* moiety yields the most (S)-selective (96% ee) ATHase reported to date. X-ray structural data indicate that the high enantioselectivity results from embedding the piano stool moiety within the protein, consistent with the computational model.

Artificial metalloenzymes (ArMs), which combine an abiotic metal cofactor with a protein scaffold, catalyze various synthetically useful transformations. To complement the natural enzymes' repertoire, effective optimization protocols to improve ArM's performance are required. Here we report on our efforts to optimize the activity of an artificial transfer hydrogenase (ATHase) using E. coli whole cells. For this purpose, we rely on a self-immolative quinolinium substrate which, upon reduction, releases fluorescent umbelliferone, thus allowing efficient screening. Introduction of a loop in the immediate proximity of the Ircofactor afforded an ArM with up to fivefold increase in transfer hydrogenation activity compared to the wild-type ATHase using purified mutants.

Reversible-deactivation radical polymerizations (controlled radical polymerizations) have revolutionized and revitalized the field of polymer synthesis. While enzymes and other biologically derived catalysts have long been known to initiate free radical polymerizations, the ability of peroxidases, hemoglobin, laccases, enzyme-mimetics, chlorophylls, heme, red blood cells, bacteria, and other biocatalysts to control or initiate reversible-deactivation radical polymerizations has only been described recently. Here, the scope of biocatalytic atom transfer radical polymerizations (bioATRP), enzyme-initiated reversible addition-fragmentation chain transfer radical polymerizations (bioRAFT), biocatalytic organometallic mediated radical polymerizations (bioOMRP), and biocatalytic reversible complexation mediated polymerizations (bioRCMP) is critically reviewed and the potential of these reactions for the environmentally friendly synthesis of precision polymers, for the preparation of functional nanostructures, for the modification of surfaces, and for biosensing is discussed. Biologically derived catalysts, such as enzymes or their cofactors, represent an attractive alternative to conventional polymerization catalysts because they are non-toxic, biodegradable, and derived from sustainable resources. Moreover, enzymes can display high stereo-, regio-, or chemo-selectivity, 1 while working under mild conditions. They have been extensively explored for the in vitro synthesis of polymers, 1-3 e.g. by ring opening polymerization (ROP) 4-5 or polycondensation. 6-7 Several enzymes can also mediate free radical polymerizations. 8-9 For example, laccases use oxygen to create radicals on phenols, which then undergo radical coupling polymerization. 10 This reaction has been used since ancient times to create traditional Japanese lacquerware from the sap of the lacquer tree Rhus vernicifera that contains the monomers and the enzyme. 11 Enzymatic radical polymerizations are also involved in the biosynthesis of lignin 12 and of melanin. 13 Not surprisingly, radicalproducing enzymes have also been explored in synthetic polymer chemistry, for example to polymerize vinyl monomers (e.g. acrylates and acrylamides), 8, 14 anilines, 15 phenols 2, 16-17 and lignols. 3, 18-19 While peroxidases and other heme proteins, as well as laccases, can initiate free radical polymerizations using peroxides and oxygen, respectively, 8-9 until recently it was unknown that biocatalysts can also control or initiate radical polymerizations in very similar ways to conventional catalysts for reversible-deactivation radical polymerizations (also termed controlled radical polymerizations (CRPs). 20-23 Here, we review the nascent field of biocatalytic controlled radical polymerizations (bioCRP) and critically discuss the potential of these novel enzymatic polymerizations in applications such as polymer synthesis, development of functional nanostructures, and biosensing.