Selective Oxidations Using a Cytochrome P450 Enzyme Variant Driven with Surrogate Oxygen Donors and Light

Abstract

Cytochrome P450 monooxygenase enzymes are versatile catalysts, which have been adapted for multiple applications in chemical synthesis. Mutation of a highly conserved active site threonine to a glutamate can convert these enzymes into peroxygenases that utilise hydrogen peroxide (H2O2). Here, we use the T252E‐CYP199A4 variant to study peroxide‐driven oxidation activity by using H2O2 and urea‐hydrogen peroxide (UHP). We demonstrate that the T252E variant has a higher stability to H2O2 in the presence of substrate that can undergo carbon‐hydrogen abstraction. This peroxygenase variant could efficiently catalyse O‐demethylation and an enantioselective epoxidation reaction (94 % ee). Neither the monooxygenase nor peroxygenase pathways of the P450 demonstrated a significant kinetic isotope effect (KIE) for the oxidation of deuterated substrates. These new peroxygenase variants offer the possibility of simpler cytochrome P450 systems for selective oxidations. To demonstrate this, a light driven H2O2 generating system was used to support efficient product formation with this peroxygenase enzyme.

Efficient regio- and stereo-selective C–H bond hydroxylation of steroids using an engineered heme-thiolate peroxygenase biocatalyst

“…Positive cooperativity was observed for progesterone binding but not androstenedione (see ESI, † Table S2). The lower binding affinity of the variant may simply be a reflection of a greater degree of difficulty in removing the 6th aqua ligand in these P450 variants, as has been reported with other enzymes, 19 rather than an intrinsic weaker binding of the steroid within the active site.…”

“…The result with the T258E mutant was somewhat surprising as the equivalent mutation in other cytochrome P450 enzymes has been demonstrated to prevent the displacement of the 6th aqua ligand on substrate binding. 13,18,19 The binding affinity of both progesterone and androstenedione was measured with the WT and T258E variants of CYP154C8 (Fig. 3 and S3 †).…”

“…S9 †). 19,22 The oxidation of androstenedione was also assessed with the T258E variant of CYP154C8. This substrate was also oxidised to a single major metabolite (Fig.…”

“…The result with the T258E mutant was somewhat surprising as the equivalent mutation in other cytochrome P450 enzymes has been demonstrated to prevent the displacement of the 6th aqua ligand on substrate binding. 13,18,19…”

Deuterated squalene and sterols from modifiedSaccharomyces cerevisiae

“…[1][2][3][4][5] Furthermore, there is interest in deuterated molecules that display kinetic differences in activity for new pharmaceuticals and functional materials. [6][7][8][9] Although deuterated fatty acids and compounds derived therefrom are relatively freely available by metal-catalysed H/D exchange chemistry, the same is not true for deuterated sterols and related lipids. Even cholesterol, which is approximately 30% of the composition of cell membranes and is the biosynthetic precursor to other important steroids, as well as an important component in lipid nanoparticle (LNP) technologies including mRNA vaccines, is not available commercially in a uniformly deuterated form.…”