Semi-rational engineering of CYP153A35 to enhance ω-hydroxylation activity toward palmitic acid

Abstract

CYP153A35 from Gordonia alkanivorans was recently characterized as fatty acid ω-hydroxylase. To enhance the catalytic activity of CYP153A35 toward palmitic acid, site-directed saturation mutagenesis was attempted using a semi-rational approach that combined structure-based computational analysis and subsequent saturation mutagenesis. Using colorimetric high-throughput screening (HTS) method based on O-demethylation activity of P450, CYP153A35 D131S and D131F mutants were selected. The best mutant, D131S, having a single mutation on BC-loop, showed 13- and 17-fold improvement in total turnover number (TTN) and catalytic efficiency (k /K) toward palmitic acid compared to wild-type, respectively. However, in whole-cell reaction, D131S mutant showed only 50% improvement in ω-hydroxylated palmitic acid yield compared to the wild type. Docking simulation studies explained that the effect of D131S mutation on the catalytic activity would be mainly caused by the binding pose of fatty acids in the substrate access tunnel of the enzyme. This effect of D131S mutation on the catalytic activity is synergistic with that of the mutations in the active site previously reported.

Rational Engineering ofBacillus cereusLeucine Dehydrogenase Towards α-keto Acid Reduction for Improving Unnatural Amino Acid Production

“…An improvement of hydrophobicity of substrate entrance tunnel might improve not only the catalytic efficiency but also the stability, as variant E116V showed better stability and 1.45–6.91 times higher k cat /K m than that of wild type Bc LeuDH. It was worthy to note that selection of mutational target residues should be studied for the substrate entrance tunnel as well as in the active site, as the identified residues might have synergetic effects on increasing its catalytic efficiency, which has also been reported in CYP153A family enzymes . The spatial conformation of the wild type Bc LeuDH substrate binding pocket was suitable for α‐keto acids without complicated branch‐chain, such as α‐ketobutyric acid and 4‐methyl‐2‐oxopentanoic acid.…”

Directed evolution methods for overcoming trade‐offs between protein activity and stability

“…The importance of rationally designing enzyme libraries with amino acid diversity in specific regions has also been recently demonstrated in a variety of examples that involve optimization of substrate access tunnels of enzyme active sites to improve activity and stability . Several computational tools have been generated for identifying and engineering substrate access tunnels for diverse applications in enzyme engineering, including altering substrate selectivity (see references within a recent review).…”

Biosynthesis of Nylon 12 Monomer, ω-Aminododecanoic Acid Using Artificial Self-Sufficient P450, AlkJ and ω-TA

“…Moreover, DDA shows relatively high solubility in water compared to other medium-to long-chain fatty acid substrates. Earlier, our group reported that CYP153A13 from Alcanivorax borkumensis SK2 efficiently transformed DDA to ω-hydroxy dodecanoic acid (ω-OHDDA) in an in vivo reaction [10][11][12].…”

“…As CYP153A belongs to the class I CYPs, three component systems are required for the activation of the enzyme ( Figure S1). Two additional proteins-putidaredoxin reductase (CamA) and putidaredoxin (CamB)-are related to electron transfer system [10][11][12]. Self-sufficient P450s, belonging to Class VIII CYPs, are catalytically self-sufficient monooxygenase that contain a heme domain (P450 domain) and a flavin reductase domain (cytochrome P450 reductase (CPR) domain) on a single polypeptide chain ( [11,12,[17][18][19]; Figure S1).…”

“…Two additional proteins-putidaredoxin reductase (CamA) and putidaredoxin (CamB)-are related to electron transfer system [10][11][12]. Self-sufficient P450s, belonging to Class VIII CYPs, are catalytically self-sufficient monooxygenase that contain a heme domain (P450 domain) and a flavin reductase domain (cytochrome P450 reductase (CPR) domain) on a single polypeptide chain ( [11,12,[17][18][19]; Figure S1). Recently published findings have shown that the fusion construct of CYP153A with the CPR domain of natural self-sufficient P450s results in the generation of the active form of the enzyme and successfully catalyzes a biocatalytic reaction [11,12,17].…”