Precise Design of Artificial Cofactors for Enhancing Peroxidase Activity of Myoglobin: Myoglobin Mutant H64D Reconstituted with a “Single-Winged Cofactor” is Equivalent to Native Horseradish Peroxidase in Oxidation Activity
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.
Recent Developments on Creation of Artificial MetalloenzymesReview
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.