Construction of biocatalysts using the myoglobin scaffold for the synthesis of indigo from indole

Hydrogen peroxide-dependent oxidation of indole producing indigo has been catalyzed by engineered myoglobins (Mbs). We have investigated a series of H64D mutants of Mbs in which Asp-64 accelerates the reaction of the Mbs with hydrogen peroxide and stabilizes the resulting active species: compound I (iron oxo-ferryl porphyrin π-cation radical). The catalytic activity of mutants depends on the amino acids at 68 and 107 positions and the H64D/V68I/I107A mutant gave the highest catalytic activity, suggesting that the side chains of Ile-68 and Ala-107 provide a hydrophobic environment suitable for indole binding. The kcat of the H64D/V68I/I107A mutant achieved 72 min−1, indicating that even Mb can be transformed into a biocatalyst.