Ward, T.R.

Top. Organomet. Chem. 2009, 10.1007/3418_2008_3

Artificial metalloenzymes can be created by incorporating an active metal catalyst precursor in a macromolecular host. When considering such artificial metalloenzymes, the first point to address is how to localize the active metal moiety within the protein scaffold. Although a covalent anchoring strategy may seem most attractive at first, supramolecular anchoring strategy has proven most successful thus far. In this context and inspired by Whitesides’ seminal paper, we have exploited the biotin–avidin technology to anchor a biotinylated active metal catalyst precursor within either avidin or streptavidin. A combined chemical and genetic strategy allows a rapid (chemogenetic) optimization of both the activity and the selectivity of the resulting artificial metalloenzymes. The chiral environment, provided by second coordination sphere interactions between the metal and the host protein, can be varied by introduction of a spacer between the biotin anchor and the metal moiety or by variation of the ligand scaffold. Alternatively, mutagenesis of the host protein allows a fine tuning of the activity and the selectivity. With this protocol, we have been able to produce artificial metalloenzymes based on the biotin–avidin technology for the enantioselective hydrogenation of N-protected dehydroaminoacids, the transfer hydrogenation of prochiral ketones as well as the allylic alkylation of symmetric substrates. In all cases selectivities >90% were achieved. Most recently, guided by an X-ray structure of an artificial metalloenzyme, we have extended the chemogenetic optimization to a designed evolution scheme. Designed evolution combines rational design with combinatorial screening. In this chapter, we emphasize the similarities and the differences between artificial metalloenzymes and their homogeneous or enzymatic counterparts.

Watanabe, Y.

Top. Organomet. Chem. 2009, 10.1007/3418_2008_4

Molecular design of artificial metalloproteins is one of the most attractive subjects in bioinorganic chemistry. Protein vacant space has been utilized to prepare metalloproteins because it provides a unique chemical environment for application to catalysts and to biomaterials bearing electronic, magnetic, and medical properties. Recently, X-ray crystal structural analysis has increased in this research area because it is a powerful tool for understanding the interactions of metal complexes and protein scaffolds, and for providing rational design of these composites. This chapter reviews the recent studies on the preparation methods and X-ray crystal structural analyses of metal/protein composites, and their functions as catalysts, metal-drugs, etc.

DeGrado, W.F.

Nat. Rev. Chem. 2022, 6, 31-50, 10.1038/s41570-021-00339-5

Natural metalloproteins perform many functions — ranging from sensing to electron transfer and catalysis — in which the position and property of each ligand and metal are dictated by protein structure. De novo protein design aims to define an amino acid sequence that encodes a specific structure and function, providing a critical test of the hypothetical inner workings of (metallo)proteins. To date, de novo metalloproteins have used simple, symmetric tertiary structures — uncomplicated by the large size and evolutionary marks of natural proteins — to interrogate structure–function hypotheses. In this Review, we discuss de novo design applications, such as proteins that induce complex, increasingly asymmetric ligand geometries to achieve function, as well as the use of more canonical ligand geometries to achieve stability. De novo design has been used to explore how proteins fine-tune redox potentials and catalyse both oxidative and hydrolytic reactions. With an increased understanding of structure–function relationships, functional proteins including O2-dependent oxidases, fast hydrolases and multi-proton/multielectron reductases have been created. In addition, proteins can now be designed using xenobiological metals or cofactors and principles from inorganic chemistry to derive new-to-nature functions. These results and the advances in computational protein design suggest a bright future for the de novo design of diverse, functional metalloproteins.

Reetz, M.T.

Top. Organomet. Chem. 2009, 10.1007/3418_2008_12

Whereas the directed evolution of stereoselective enzymes provides a useful tool in asymmetric catalysis, generality cannot be claimed because enzymes as catalysts are restricted to a limited set of reaction types. Therefore, a new concept has been proposed, namely directed evolution of hybrid catalysts in which proteins serve as hosts for anchoring ligand/transition metal entities. Accordingly, appropriate genetic mutagenesis methods are applied to the gene of a given protein host, providing after expression a library of mutant proteins. These are purified and a ligand/transition metal anchored site-specifically. Following en masse ee-screening, the best hit is identified, and the corresponding mutant gene is used as a template for another round of mutagenesis, expression, purification, bioconjugation, and screening. This allows for a Darwinian optimization of transition metal catalysts.

Kazlauskas, R.J.

Top. Organomet. Chem. 2009, 10.1007/3418_2008_1

Carbonic anhydrase binds a zinc ion in a hydrophobic active site using the imidazole groups of three histidine residues. The natural role of carbonic anhydrase is to catalyze the reversible hydration of carbon dioxide to bicarbonate, but it also catalyzes hydrolysis of esters with moderate enantioselectivity. Replacing the active-site zinc with manganese yielded manganese-substituted carbonic anhydrase (CA[Mn]), which shows peroxidase activity with a bicarbonate-dependent mechanism. In the presence of bicarbonate and hydrogen peroxide, CA[Mn] catalyzed the efficient oxidation of o-dianisidine with k cat /K M = 1.4 × 106 M−1s−1, which is comparable to that for horseradish peroxidase, k cat /K M = 57 × 106 M−1s−1. CA[Mn] also catalyzed the moderately enantioselective epoxidation of olefins to epoxides (E = 5 for p-chlorostyrene). This enantioselectivity is similar to that for natural heme-based peroxidases, but has the advantage that CA[Mn] avoids formation of aldehyde side products. CA[Mn] degrades during the epoxidation, limiting the yield of the epoxidations to <12%. Replacement of active-site residues Asn62, His64, Asn67, Gln92, or Thr200 with alanine by site-directed mutagenesis decreased the enantioselectivity showing that the active site controls enantioselectivity of the epoxidation.

Sheldon, R.A.

Can. J. Chem. 2002, 80, 622-625, 10.1139/v02-082

The catalytic Zn2+ ion was extracted from thermolysin, which had been covalently bound to Eupergit C. The apo-enzyme incorporated the oxometallate anions MoO42–, SeO42–, and WO42– with partial restoration of the proteolytic activity. Tungstate thermolysin was moderately active in the sulfoxidation of thioanisole by hydrogen peroxide, whereas its activity towards phenylmercaptoacetophenone, which was designed to bind well in the active site of thermolysin, was much higher.

Mayer, C.; Roelfes, G.

Nat. Rev. Chem. 2019, 3, 687-705, 10.1038/s41570-019-0143-x

The ability of one enzyme to catalyse multiple, mechanistically distinct transformations likely played a crucial role in organisms’ abilities to adapt to changing external stimuli in the past and can still be observed in extant enzymes. Given the importance of catalytic promiscuity in nature, enzyme designers have recently begun to create catalytically promiscuous enzymes in order to expand the canon of transformations catalysed by proteins. This article aims to both critically review different strategies for the design of enzymes that display catalytic promiscuity for new-to-nature reactions and highlight the successes of subsequent directed-evolution efforts to fine-tune these novel reactivities. For the former, we put a particular emphasis on the creation, stabilization and repurposing of reaction intermediates, which are key for unlocking new activities in an existing or designed active site. For the directed evolution of the resulting catalysts, we contrast approaches for enzyme design that make use of components found in nature and those that achieve new reactivities by incorporating synthetic components. Following the critical analysis of selected examples that are now available, we close this Review by providing a set of considerations and design principles for enzyme engineers, which will guide the future generation of efficient artificial enzymes for synthetically useful, abiotic transformations.

Happe, T.; Hemschemeier, A.

Nat. Rev. Chem. 2018, 2, 231-243, 10.1038/s41570-018-0029-3

Metal cofactors considerably widen the catalytic space of naturally occurring enzymes whose specific and enantioselective catalytic activity constitutes a blueprint for economically relevant chemical syntheses. To optimize natural enzymes and uncover novel reactivity, we need a detailed understanding of cofactor–protein interactions, which can be challenging to obtain in the case of enzymes with sophisticated cofactors. As a case study, we summarize recent research on the [FeFe]-hydrogenases, which interconvert protons, electrons and dihydrogen at a unique iron-based active site. We can now chemically synthesize the complex cofactor and incorporate it into an apo-protein to afford functional enzymes. By varying both the cofactor and the polypeptide components, we have obtained detailed knowledge on what is required for a metal cluster to process H2. In parallel, the design of artificial proteins and catalytically active nucleic acids are advancing rapidly. In this Perspective, we introduce these fields and outline how chemists and biologists can use this knowledge to develop novel tailored semisynthetic catalysts.