Structural Insights, Catalytic Mechanism, and Tunnel Engineering of the Multifunctional Cytochrome P450 DoxA

The multifunctional cytochrome P450 DoxA, central to anthracycline biosynthesis, demonstrates unprecedented catalytic versatility by performing both 13- and 14-hydroxylation of daunorubicin precursors to form doxorubicin (DXR). Structural elucidation (2.8 Å resolution) reveals a classic P450 fold with an open, flexible substrate-binding pocket and six substrate recognition sites, enabling steric accommodation of bulky intermediates. Remarkably, DoxA employs three distinct catalytic strategies: (1) conventional redox protein-dependent electron transfer, (2) direct NADPH-mediated H₂O₂ generation driving peroxygenase-like activity, and (3) exogenous H₂O₂ utilization, with hydroxylation efficiencies scaling with peroxide concentration. Tunnel engineering targeting H₂O₂ access pathways yielded mutants (e.g., R303G) with 4-fold enhanced activity at low H₂O₂ (5 mM), mitigating oxidative damage. This work establishes DoxA as a paradigm of P450 mechanistic adaptability, combining structural plasticity with multi-pathway catalysis, while its engineered variants offer biotechnological potential for streamlined DXR production.