Efficient hydroxylation of cycloalkanes by co-addition of decoy molecules to variants of the cytochrome P450 CYP102A1.

The wild-type cytochrome P450 (CYP) monooxygenase enzyme CYP102A1 (P450Bm3) has low activity for cycloalkane oxidation. The oxidation of these substrates by variants of this enzyme in combination with perfluorinated decoy molecules (PFCs) was investigated to improve productivity. The use of rate accelerating variants, which have mutations located outside of the substrate binding pocket as well as an active site variant of CYP102A1 (A74G/F87V/L188Q) all enhanced cycloalkane oxidation (C5 to C10). The addition of the decoy molecules to the wild-type and the rate accelerating mutants of CYP102A1 boosted the substrate oxidation rates even further. However, the levels of cycloalkanol product decreased with the larger alkanes when the decoy molecules were used with the variant A74G/F87V/L188Q, which contained mutations within the substrate binding pocket. For the majority of the enzymes and PFC decoy molecule combinations the highest levels of oxidation were obtained with cyclooctane. When larger second generation decoy molecules, based on modified amino acids were utilised there was a significant improvement in the oxidation of the smaller cycloalkanes by the wild-type enzyme and one other variant. This resulted in significant improvements in biocatalytic oxidation of cyclopentane and cyclohexane. However, the use of these optimised decoy molecules did not significantly improve cycloalkane oxidation over the fluorinated fatty acid derivatives when combined with the best rate accelerating variant, R47L/Y51F/I401P. Overall our approach enabled the cycloalkanes to be oxidised 300- to 8000-fold more efficiently than the wild-type enzyme at product formation rates in excess of 500 and up to 1700 nmol·nmol-CYP-1·min-1.

Stereoselective hydroxylation of isophorone by variants of the cytochromes P450 CYP102A1 and CYP101A1.

The stereoselective oxidation of hydrocarbons is an area of research where enzyme biocatalysis can make a substantial impact. The cyclic ketone isophorone was stereoselectively hydroxylated (≥95%) by wild-type CYP102A1 to form (R)-4-hydroxyisophorone, an important chiral synthon and flavour and fragrance compound. CYP102A1 variants were also selective for 4-hydroxyisophorone formation and the product formation rate increased over the wild-type enzyme by up to 285-fold, with the best mutants being R47L/Y51F/I401P and A74G/F87V/L188Q. The latter variant, which contained mutations in the distal substrate binding pocket, was marginally less selective. Combining perfluorodecanoic acid decoy molecules with the rate accelerating variant R47L/Y51F/I401P engendered further improvement with the purified enzymes. However when the decoy molecules were used with A74G/F87V/L188Q the amount of product generated by the enzyme was reduced. Addition of decoy molecules to whole-cell turnovers did not improve the productivity of these CYP102A1 systems. WT CYP101A1 formed significant levels of 7-hydroxyisophorone as a minor product alongside 4-hydroxyisophorone. However the F87W/Y96F/L244A/V247L CYP101A1 mutant was ≥98% selective for (R)-4-hydroxyisophorone. A comparison of the two enzyme systems using whole-cell oxidation reactions showed that the best CYP101A1 variant was able to generate more product. We also characterised that the further oxidation metabolite 4-ketoisophorone was produced and then subsequently reduced to levodione by an endogenous Escherichia coli ene reductase.