Enhancing the activity of the bifunctional membrane Δ12-hydroxylase/desaturase CpFAH12: Key insights from redox partner interactions and electron transfer pathways.
TL;DR
Using the best-characterized mutant, ricinoleic acid production in Erlenmeyer flasks reached 3.8 g/L, representing nearly a two-fold increase compared to best reported titers to date, while providing deeper insights into the molecular determinants governing the activity and specificity of membrane-bound desaturases.
Abstract
Fatty acid desaturases introduce double bonds into fatty acyl chains to produce unsaturated fatty acids. Some membrane-bound desaturases also catalyze hydroxylation, yet the mechanistic link between desaturation and hydroxylation in bifunctional enzymes remains unresolved. Here, we combined molecular modelling and experimental validation to investigate the monofunctional desaturase CpFAD2 and the bifunctional desaturase/hydroxylase CpFAH12 from Claviceps purpurea. Structural models of CpFAH12 and CpFAD2, bound to their oleoyl-phosphatidylcholine substrate and cytochrome b₅ redox partner, revealed highly similar catalytic and substrate-binding sites. Analysis of enzyme-cytochrome b₅ interactions showed that these are primarily stabilized by electrostatic interactions in the cytosolic domain and hydrophobic contacts within the transmembrane regions. Notably, residues R359 and R399 in CpFAH12 play a critical role in stabilizing the CpFAH12/cytochrome b₅ complex via electrostatic interactions absent in CpFAD2. Directed mutagenesis at adjacent position 358 (replacing CpFAH12 residues with those from CpFAD2) enhanced overall enzyme activity and hydroxylation efficiency. Using the best-characterized mutant, ricinoleic acid production in Erlenmeyer flasks reached 3.8 g/L, representing nearly a two-fold increase compared to best reported titers to date using wild-type CpFAH12, while providing deeper insights into the molecular determinants governing the activity and specificity of membrane-bound desaturases.