Introduction of aromatic amino acids in electron transfer pathways yielded improved catalytic performance of cytochrome P450s

Abstract

Cytochrome P450s are versatile catalysts for biosynthesis applications. In the P450 catalytic cycle, two electrons are required to reduce the heme iron and activate the subsequent reductions through proposed electron transfer pathways (eTPs), which often represent the rate-limiting step in reactions. Herein, the P450 BM3 from Bacillus megaterium was engineered for improved catalytic performance by redesigning proposed eTPs. By introducing aromatic amino acids on eTPs of P450 BM3, the “best” variant P2H02 (A399Y/Q403F) showed 13.9-fold improved catalytic efficiency (kcat/KM= 913.5 L mol−1 s−1) compared with P450 BM3 WT (kcat/KM= 65.8 L mol−1 s−1). Molecular dynamics simulations and electron hopping pathways analysis revealed that aromatic amino acid substitutions bridging the cofactor flavin mononucleotide and heme iron could increase electron transfer rates and improve catalytic performance. Moreover, the introduction of tyrosines showed positive effects on catalytic efficiency by potentially protecting P450 from oxidative damage. In essence, engineering of eTPs by aromatic amino acid substitutions represents a powerful approach to design catalytically efficient P450s (such as CYP116B3) and could be expanded to other oxidoreductases relying on long-range electron transfer pathways.