Abstract
Cytochrome P450 CYP153AM.aq from Marinobacter aquaeolei serves as a model enzyme for the terminal (ω-) hy-droxylation of medium- to long-chain fatty acids. We have engineered this enzyme using different mutagenesis approaches based on structure-sequence-alignments within the 3DM database, crystal structures of CYP153M.aq. and a homolog CYP153AP.sp and site-directed saturation mutagenesis, to create a variant that ω-hydroxylates octanoic acid. The M.aqRLT variant exhibited 151-fold improved catalytic efficiency and showed strongly improved substrate binding, indicated by a 25-fold reduced Km compared to the wild type. Hence, we further investigated the variant computationally using MD simulations to gain deeper insights into the dynamics of the protein. We found the tunnel modifications and the two loop regions showing greatly reduced flexibility in the new variant were the main features responsible for stabilizing the ligand in the active site and enhancing catalytic efficiency. Additionally, we showed that a previously known fatty acid anchor (Q129R) interacts significantly with the ligand to hold it in the reactive position, thereby boosting the activity of the variant M.aq RLT towards octanoic acid. The study retrospectively demonstrates the drastic effects of substrate stabilization and simultaneously the impact of enzyme flexibility. These results could guide future engineering of enzymes with deeply buried active sites to increase or even establish new substrate activities.
Original language | English |
---|---|
Pages (from-to) | 3182-3189 |
Number of pages | 8 |
Journal | ACS Catalysis |
Volume | 11 |
Early online date | 24 Feb 2021 |
DOIs | |
Publication status | E-pub ahead of print - 24 Feb 2021 |