Fig. 4: Optimization of CYP260A1 via redox partner screening and computational-guided enzyme engineering.
From: Discovery and engineering of bacterial P450s for C-14 hydroxylation in ent-kaurane diterpenoids
a Redox partner screening. CYP260A1 activity was assessed with different redox partners, showing CamA/CamB significantly enhanced product yield compared to the original RhFRed system. Data are presented as mean ± SD (n = 3, biological replicates). b Active site analysis. MM/PBSA analysis identifies residues within 4 Å of CYP260A1’s active site that negatively impact substrate binding. c Energy contribution analysis with Poisson-Boltzmann (PB). Residues with positive values negatively impacted catalysis, while those with negative values improved substrate binding. d Experimental validation of mutations. The L162V variant exhibited the highest production titer, increasing product yield to 84.2 ± 10.3 mg/L, an 8.3-fold improvement over the wild-type. Error bars indicate the standard deviation of three independent biological replicates. Statistical analysis was performed using a one-way ANOVA across all genotypes, followed by Tukey’s two-sided multiple-comparisons test. e Active-site view highlighting two key variants (L162V and A74L) of CYP260A1, with blue and red denoting the side chains of the wild-type and mutated residues, respectively. f Distance between the C-14 atom of substrate 1 and the heme iron center over a 100 ns MD simulation. The black, blue, and red traces represent the distances for wild-type, A74L, and L162V, respectively. Source data are provided as a Source Data file.
