An In Crystallo Reaction with an Engineered Cytochrome P450 Peroxygenase.

The cytochrome P450 monooxygenases (CYPs) are a class of heme-thiolate enzymes that insert oxygen into unactivated C-H bonds. These enzymes can be converted into peroxygenases via protein engineering, which enables their activity to occur using hydrogen peroxide (H2 O2 ) without the requirement for additional nicotinamide co-factors or partner proteins. Here, we demonstrate that soaking crystals of an engineered P450 peroxygenase with H2 O2 enables the enzymatic reaction to occur within the crystal. Crystals of the designed P450 peroxygenase, the T252E mutant of CYP199A4, in complex with 4-methoxybenzoic acid were soaked with different concentrations of H2 O2 for varying times to initiate the in crystallo O-demethylation reaction. Crystal structures of T252E-CYP199A4 showed a distinct loss of electron density that was consistent with the O-demethylated metabolite, 4-hydroxybenzoic acid. A new X-ray crystal structure of this enzyme with the 4-hydroxybenzoic acid product was obtained to enable comparison alongside the existing substrate-bound structure. The visualisation of enzymatic catalysis in action is challenging in structural biology and the ability to initiate the reactions of P450 enzymes, in crystallo by simply soaking crystals with H2 O2 will enable new structural biology methods and techniques to be applied to study their mechanism of action.

Engineering C-C Bond Cleavage Activity into a P450 Monooxygenase Enzyme.

The cytochrome P450 (CYP) superfamily of heme monooxygenases has demonstrated ability to facilitate hydroxylation, desaturation, sulfoxidation, epoxidation, heteroatom dealkylation, and carbon-carbon bond formation and cleavage (lyase) reactions. Seeking to study the carbon-carbon cleavage reaction of α-hydroxy ketones in mechanistic detail using a microbial P450, we synthesized α-hydroxy ketone probes based on the physiological substrate for a well-characterized benzoic acid metabolizing P450, CYP199A4. After observing low activity with wild-type CYP199A4, subsequent assays with an F182L mutant demonstrated enzyme-dependent C-C bond cleavage toward one of the α-hydroxy ketones. This C-C cleavage reaction was subject to an inverse kinetic solvent isotope effect analogous to that observed in the lyase activity of the human P450 CYP17A1, suggesting the involvement of a species earlier than Compound I in the catalytic cycle. Co-crystallization of F182L-CYP199A4 with this α-hydroxy ketone showed that the substrate bound in the active site with a preference for the (S)-enantiomer in a position which could mimic the topology of the lyase reaction in CYP17A1. Molecular dynamics simulations with an oxy-ferrous model of CYP199A4 revealed a displacement of the substrate to allow for oxygen binding and the formation of the lyase transition state proposed for CYP17A1. This demonstration that a correctly positioned α-hydroxy ketone substrate can realize lyase activity with an unusual inverse solvent isotope effect in an engineered microbial system opens the door for further detailed biophysical and structural characterization of CYP catalytic intermediates.

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

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 (H2 O2 ). Here, we use the T252E-CYP199A4 variant to study peroxide-driven oxidation activity by using H2 O2 and urea-hydrogen peroxide (UHP). We demonstrate that the T252E variant has a higher stability to H2 O2 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 H2 O2 generating system was used to support efficient product formation with this peroxygenase enzyme.

The Stereoselective Oxidation of para-Substituted Benzenes by a Cytochrome P450 Biocatalyst.

The serine 244 to aspartate (S244D) variant of the cytochrome P450 enzyme CYP199A4 was used to expand its substrate range beyond benzoic acids. Substrates, in which the carboxylate group of the benzoic acid moiety is replaced were oxidised with high activity by the S244D mutant (product formation rates >60 nmol.(nmol-CYP)-1 .min-1 ) and with total turnover numbers of up to 20,000. Ethyl α-hydroxylation was more rapid than methyl oxidation, styrene epoxidation and S-oxidation. The S244D mutant catalysed the ethyl hydroxylation, epoxidation and sulfoxidation reactions with an excess of one stereoisomer (in some instances up to >98 %). The crystal structure of 4-methoxybenzoic acid-bound CYP199A4 S244D showed that the active site architecture and the substrate orientation were similar to that of the WT enzyme. Overall, this work demonstrates that CYP199A4 can catalyse the stereoselective hydroxylation, epoxidation or sulfoxidation of substituted benzene substrates under mild conditions resulting in more sustainable transformations using this heme monooxygenase enzyme.

Selective hydroxylation of 1,8- and 1,4-cineole using bacterial P450 variants.

This study has evaluated the use of the P450 metalloenzymes CYP176A1, CYP101A1 and CYP102A1, together with engineered protein variants of CYP101A1 and CYP102A1, to alter the regioselectivity of 1,8- and 1,4-cineole hydroxylation. CYP176A1 was less selective for 1,4-cineole oxidation when compared to its preferred substrate, 1,8-cineole. The CYP102A1 variants significantly improved the activity over the WT enzyme for oxidation of 1,4- and 1,8-cineole. The CYP102A1 R47L/Y51F/A74G/F87V/L188Q mutant generated predominantly (1S)-6α-hydroxy-1,8-cineole (78% e.e.) from 1,8-cineole. Oxidation of 1,4-cineole by the CYP102A1 R47L/Y51F/F87A/I401P variant generated the 3α product in >90% yield. WT CYP101A1 formed a mixture metabolites with 1,8-cineole and very little product was generated with 1,4-cineole. In contrast the F87W/Y96F/L244A/V247L and F87W/Y96F/L244A variants of CYP101A1 favoured formation of 5α-hydroxy-1,8-cineole (>88%, 1S 86% e.e.) while the F87V/Y96F/L244A variant generated (1S)-6α-hydroxy-1,8-cineole in excess (90% regioselective, >99% e.e.). The CYP101A1 F87W/Y96F/L244A/V247L and F87W/Y96F/L244A mutants improved the oxidation of 1,4-cineole generating an excess of the 3α metabolite (1S > 99% e.e. with the latter). The CYP101A1 F87L/Y96F variant also improved the oxidation of this substrate but shifted the site of oxidation to the isopropyl group, (8-hydroxy-1,4-cineole). When this 8-hydroxy metabolite was generated in significant quantities desaturation of C8C9 to the corresponding alkene was also detected.

Biomimetic and Biocatalytic Synthesis of Bruceol.

The first total synthesis of bruceol has been achieved using a biomimetic cascade cyclization initiated by a stereoselective Jacobsen-Katsuki epoxidation (and kinetic resolution) of racemic protobruceol-I. A bacterial cytochrome P450 monooxygenase was also found to catalyze the conversion of protobruceol-I into bruceol. The first full analysis of the NMR data of natural bruceol suggested that "isobruceol" was a previously unrecognized natural product also isolated from Philotheca brucei. This was confirmed by the re-isolation, synthesis, and X-ray analysis of isobruceol. In total, eight stereoisomers and structural isomers of bruceol have been synthesized in a highly divergent approach.

The Oxidation of Hydrophobic Aromatic Substrates by Using a Variant of the P450 Monooxygenase CYP101B1.

The cytochrome P450 monooxygenase CYP101B1, from a Novosphingobium bacterium is able to bind and oxidise aromatic substrates but at a lower activity and efficiency than norisoprenoids and monoterpenoid esters. Histidine 85 of CYP101B1 aligns with tyrosine 96 of CYP101A1, which, in the latter enzyme forms the only hydrophilic interaction with its substrate, camphor. The histidine residue of CYP101B1 was mutated to phenylalanine with the aim of improving the activity of the enzyme for hydrophobic substrates. The H85F mutant lowered the binding affinity and activity of the enzyme for ß-ionone and altered the oxidation selectivity. This variant also showed enhanced affinity and activity towards alkylbenzenes, styrenes and methylnaphthalenes. For example the rate of product formation for acenaphthene oxidation was improved sixfold to 245 nmol per nmol CYP per min. Certain disubstituted naphthalenes and substrates, such as phenylcyclohexane and biphenyls, were oxidised with lower activity by the H85F variant. Variants at H85 (A and G) designed to introduce additional space into the active site so as to accommodate these larger substrates did not improve the oxidation activity. As the H85F mutant of CYP101B1 improved the oxidation of hydrophobic substrates, this residue is likely to be in the substrate binding pocket or the access channel of the enzyme. The side chain of the histidine might interact with the carbonyl groups of the favoured norisoprenoid substrates of CYP101B1.

Selective carbon-hydrogen bond hydroxylation using an engineered cytochrome P450 peroxygenase.

The cytochrome P450 enzyme CYP102A1 (P450BM3) is a versatile monooxygenase enzyme which has been adapted and engineered for multiple applications in chemical synthesis. Mutation of threonine 268 to glutamate (Thr268Glu) converted the heme domain of this enzyme into a H2O2 utilizing peroxygenase. This variant displayed significantly increased peroxide driven hydroxylation activity towards the saturated linear fatty acids tested (undecanoic through to hexadecenoic acid) when compared to the wild-type heme domain. The product distributions arising from fatty acid oxidation using this peroxygenase variant were broadly similar to those obtained with the wild-type monooxygenase holoenzyme, with oxidation occurring predominantly at the ω-1 through to ω-3 positions. 10-Undecenoic acid was regioselectively hydroxylated at the allylic ω-2 carbon by the Thr268Glu peroxygenase. The effect of isotopic substitution were measured using [9,9,10,10-d4]-dodecanoic acid. The kinetic isotope effect for both the monooxygenase and peroxygenase systems ranged between 7.9 and 9.5, with that of the peroxygenase enzyme being marginally lower. This highlights that carbon­hydrogen bond abstraction is important in the mechanism of both the monooxygenase and peroxygenase systems. This would infer that the ferryl-oxo radical cation intermediate, compound I, is the likely reactive intermediate in both systems. The peroxygenase variant offers the possibility of simpler cytochrome P450 systems for selective oxidations. To demonstrate this we used this system to oxidize tetradecanoic acid using light driven generation of H2O2 by a flavin.

Characterisation of the heme aqua-ligand coordination environment in an engineered peroxygenase cytochrome P450 variant.

The cytochrome P450 enzymes (CYPs) are heme-thiolate monooxygenases that catalyse the insertion of an oxygen atom into the C-H bonds of organic molecules. In most CYPs, the activation of dioxygen by the heme is aided by an acid-alcohol pair of residues located in the I-helix of the enzyme. Mutation of the threonine residue of this acid-alcohol pair of CYP199A4, from the bacterium Rhodospeudomonas palustris HaA2, to a glutamate residue induces peroxygenase activity. In the X-ray crystal structures of this variant an interaction of the glutamate side chain and the distal aqua ligand of the heme was observed and this results in this ligand not being readily displaced in the peroxygenase mutant on the addition of substrate. Here we use a range of bulky hydrophobic and nitrogen donor containing ligands in an attempt to displace the distal aqua ligand of the T252E mutant of CYP199A4. Ligand binding was assessed by UV-visible absorbance spectroscopy, native mass spectrometry, electron paramagnetic resonance and X-ray crystallography. None of the ligands tested, even the nitrogen donor ligands which bind directly to the iron in the wild-type enzyme, resulted in displacement of the aqua ligand. Therefore, modification of the I-helix threonine residue to a glutamate residue results in a significant strengthening of the ferric distal aqua ligand. This ligand was not displaced using any of the ligands during this study and this provides a rationale as to why this mutant can shutdown the monooxygenase pathway of this enzyme and switch to peroxygenase activity.

Exploring the Factors which Result in Cytochrome P450 Catalyzed Desaturation Versus Hydroxylation.

The cytochrome P450 family of monooxygenase enzymes have essential biological roles involving the selective oxidation of carbon-hydrogen bonds. They can also catalyze other important metabolic reactions including desaturation to form alkenes. Currently the factors that control the partition between P450 hydroxylation and desaturation pathways are poorly defined. The CYP199A4 enzyme from the bacterium Rhodopseudomonas palustris HaA2 catalyzes the oxidation of 4-ethyl- and 4-isopropyl- benzoic acids with hydroxylation and desaturation occurring in significant quantities. Here we demonstrate that 4-cyclopropylbenzoic acid is regioselectively hydroxylated by CYP199A4 at the benzylic carbon. In contrast, the oxidation of 4-n-propylbenzoic acid by CYP199A4 results in three major metabolites: an alkene from desaturation and two hydroxylation products at the benzylic (Cα) and Cß carbons in similar quantities. Extending the length of the alkyl substituent resulted in 4-n-butylbenzoic acid being oxidized at the benzylic position (45%) and desaturated (55%). In contrast, 4-isobutylbenzoic generated very little alkene (5%) but was hydroxylated at the benzylic position (54%) and at the tertiary Cß position (41%). The oxidation of 4-n-propylbenzoic acid by the F298 V mutant of CYP199A4 occurred with no hydroxylation at Cß and a significant increase in metabolites arising from desaturation (73%). The X-ray crystal structures of CYP199A4 with each substrate revealed that they bind in the active site with the alkyl substituent positioned over the heme. However, the longer alkylbenzoic acids were bound in a different conformation as was 4-n-propylbenzoic acid in the F298 V mutant. Overall, the changes in metabolite distribution could be ascribed to bond strength differences and the position of the alkyl group relative to the heme.

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.