Marchi-Delapierre, C.

ACS Catal. 2020, 10, 5631-5645, 10.1021/acscatal.9b04904

Artificial enzymes represent an attractive alternative to design abiotic biocatalysis. EcNikA-Ru1, an artificial metalloenzyme developed by embedding a ruthenium-based catalyst into the cavity of the periplasmic nickel-binding protein NikA, was found to efficiently and selectively transform certain alkenes. The objective of this study was to provide a rationale on the enzymatic function and the unexpected substrate-dependent chemoselectivity of EcNikA-Ru1 thanks to a dual experimental/computational study. We observed that the de novo active site allows the formation of the terminal oxidant via the formation of a ruthenium aquo species that subsequently reacts with the hypervalent iodine of phenyl iodide diacetic acid. The oxidation process relies on a RuIV═O pathway via a two-step reaction with a radical intermediate, resulting in the formation of either a chlorohydrin or an epoxide. The results emphasize the impact of the protein scaffold on the kinetics of the reaction, through (i) the promotion of the starting oxidizing species via the exchange of a CO ligand with a water molecule; and (ii) the control of the substrate orientation on the intermediate structures, formed after the RuIV═O attack. When a Cα attack is preferred, chlorohydrins are formed while an attack on Cβ leads to an epoxide. This work provides evidence that artificial enzymes mimic the behavior of their natural counterparts.

Matsuo, T.

Chem. - Asian J. 2011, 6, 2491-2499, 10.1002/asia.201100107

H64D myoglobin mutant was reconstituted with two different types of synthetic hemes that have aromatic rings and a carboxylate‐based cluster attached to the terminus of one or both of the heme‐propionate moieties, thereby forming a “single‐winged cofactor” and “double‐winged cofactor,” respectively. The reconstituted mutant myoglobins have smaller Km values with respect to 2‐methoxyphenol oxidation activity relative to the parent mutant with native heme. This suggests that the attached moiety functions as a substrate‐binding domain. However, the kcat value of the mutant myoglobin with the double‐winged cofactor is much lower than that of the mutant with the native heme. In contrast, the mutant reconstituted with the single‐winged cofactor has a larger kcat value, thereby resulting in overall catalytic activity that is essentially equivalent to that of the native horseradish peroxidase. Enhanced peroxygenase activity was also observed for the mutant myoglobin with the single‐winged cofactor, thus indicating that introduction of an artificial substrate‐binding domain at only one of the heme propionates in the H64D mutant is the optimal engineering strategy for improving the peroxidase activity of myoglobin.

Matsuo, T.

Tetrahedron Lett. 2019, 60, 151226, 10.1016/j.tetlet.2019.151226

Organic synthesis using biocatalysts has been developed over many years and is still a prominent area of research. In this context, various hybrid biocatalysts composed of a synthetic metal complex catalyst and a protein scaffold (i.e. artificial metalloenzymes) have been constructed. One of the most recent research areas in biocatalysts-mediated synthesis is CC bond/cleavage, the most important type of reaction in organic chemistry. Some of the artificial enzymes were applied to in-cell reactions as well as in vitro systems. The effects of the structural fluctuation in biomacromolecules on their functions have also been realized. This review article includes recent research examples of artificial metalloenzymes used to CC bond formation/cleavage. As a perspective, we also focus on how we apply protein dynamics factor for the creation of new generation artificial metalloenzymes.

Arold, S.T.; Eppinger, J.; Groll, M.

ACS Catal. 2019, 9, 11371-11380, 10.1021/acscatal.9b02896

Artificial metalloenzymes (ArMs) have high potential in biotechnological applications as they combine the versatility of transition-metal catalysis with the substrate selectivity of enzymes. An ideal host protein should allow high-yield recombinant expression, display thermal and solvent stability to withstand harsh reaction conditions, lack nonspecific metal-binding residues, and contain a suitable cavity to accommodate the artificial metal site. Moreover, to allow its rational functionalization, the host should provide an intrinsic reporter for metal binding and structural changes, which should be readily amendable to high-resolution structural characterization. Herein, we present the design, characterization, and de novo functionalization of a fluorescent ArM scaffold, named mTFP*, that achieves these characteristics. Fluorescence measurements allowed direct assessment of the scaffold’s structural integrity. Protein X-ray structures and transition metal Förster resonance energy transfer (tmFRET) studies validated the engineered metal coordination sites and provided insights into metal binding dynamics at the atomic level. The implemented active metal centers resulted in ArMs with efficient Diels–Alderase and Friedel–Crafts alkylase activities.