Hydroxylation mechanism of lignin-derived aromatic substrates catalyzed by plant P450 cinnamate 4-hydroxylase

Cytochrome P450 cinnamate 4-hydroxylase (C4H) is a pivotal enzyme in the phenylpropanoid pathway, playing a critical role in regulating lignin biosynthesis in plants. In contrast to the hydroxylation reactions catalyzed by human P450 enzymes, which have been extensively studied, the mechanistic understanding of plant P450-mediated hydroxylation of aromatic substrates remains limited. In this study, using comprehensive atomistic molecular dynamics (MD) simulations, we elucidated the binding pose of the native substrate trans-cinnamic acid and identified key residues contributing to the substrate specificity of the enzyme, which include Arg213 and a conserved hydrophobic pocket comprising Val118, Phe119, Val301, Ala302, Ile367 and Phe484. Additionally, we investigated the catalytic mechanism using hybrid quantum mechanics/molecular mechanics (QM/MM) calculations, evaluating all plausible C4H-catalysed pathways for aromatic hydroxylation. Our results reveal that among all investigated mechanisms, the most favourable pathway involves direct hydroxylation via electrophilic attack coupled with a proton shuttle. These findings provide valuable insights into the catalytic mechanism of C4H, which would pave the way for modifying lignin biosynthesis to regulate various lignin contents in plants, unlocking its potential applications in sustainable bioremediation and biomanufacturing.