Reconstitution of Myoglobin with Iron Porphycene Generates an Artificial Aldoxime Dehydratase with Expanded Catalytic Activities

Biocatalytic aldoxime dehydration is one of the most efficient methods used for the synthesis of nitriles under ambient reaction conditions without the use of highly toxic cyanides. At the same time, limitations in terms of substrate scope exist. To overcome such limitations and further extend the scope of this biocatalytic reaction, we are developing an artificial metalloenzyme with enhanced aldoxime dehydration activity based on the reconstitution of a hemoprotein. An engineered myoglobin reconstituted with an iron porphycene cofactor (rMb(FePc)) has an extremely high catalytic activity for the dehydration of diverse aldoximes. The total turnover number for the dehydration reaction reached a maximum of 9300. Furthermore, rMb(FePc) was found to promote dealcoholization of less reactive O-alkyl aldoximes which are not accepted as substrates in natural enzyme-catalyzed reactions. A series of mechanistic experiments clearly reveal the importance of hydrogen bonding between the substrates and distal His/Ser residues in the active sites. These mechanistic insights further motivated us to undertake rational mechanism-based engineering of rMb(FePc), resulting in an improved rMb(FePc) variant that is 24-fold more active than the unmutated reconstituted myoglobin catalyst.