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

Cytochromes P450 (P450s) are a family of enzymes that carry out oxidative transformations. Most commonly they catalyze the hydroxylation of carbon‑hydrogen bonds, but other more complex reactions are also supported. [1] As a result these enzymes offer advantages over synthetic methods for selective carbon‑hydrogen bond hydroxylation under mild conditions. In addition, the reactions often occur with high regio- and stereo-selectivity in a single step. [2] Most reactions catalyzed by P450 monooxygenases use the high-valent iron-oxo radical cation intermediate (compound I; Cpd I). This abstracts a hydrogen from the substrate before undergoing an oxygen rebound step (the radical rebound mechanism). [3,4] The catalytic cycle of these monooxygenase enzymes is composed of multiple steps (species I to VIII; Scheme 1) which are, in order, substrate binding, electron transfer, binding of dioxygen, and electron and proton delivery to enable O-O bond cleavage to generate Cpd I (Species VII; Scheme 1). This is followed by C-H bond abstraction and rebound to insert the oxygen atom into the organic substrate.

Protons are sourced from the bulk solvent but their delivery is controlled by specific residues within the I-helix; an acid-alcohol pair. [[5], [6], [7], [8], [9]] In most instances, the electrons are derived from a nicotinamide cofactor, NADH or NADPH, and delivered via specific electron transfer partners. [10] These reducing cofactors are expensive and attempts to replace them with simpler alternatives and do away the requirement for electron transfer proteins have been widely investigated. [[11], [12], [13], [14], [15], [16], [17]] One such method is to use hydrogen peroxide (H2O2) via the peroxide shunt mechanism (Scheme 1). [11]

Among the most studied members of the cytochrome P450 family of monooxygenases is the bacterial enzyme CYP102A1 (P450BM3) from Bacillus megaterium (recently reclassified as Prestia megaterium). [[18], [19], [20]] This P450 is relatively unusual compared to the majority of its counterparts in that its heme domain is fused to another which contains the organic electron transfer cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). This results in the enzyme being self-sufficient in that it requires no other external proteins. [19,21] It functions as a dimer, with electron transfer occurring from the FAD of one monomer to the FMN of another, and it is able to rapidly oxidize substrates such as long chain fatty acids (C12 to C16). [22,23] These are hydroxylated at multiple positions close to the omega (ω) terminus. [19] P450BM3 is easy to produce, soluble and sources its electrons from NADPH. [24] It has been modified through protein engineering techniques for the selective hydroxylation of a broad range of substrates. [25,26] Random mutagenesis and directed evolution methods have also been used to expand the substrate range of P450BM3. [[27], [28], [29], [30], [31], [32], [33], [34], [35]] In addition, it has been modified to enable non-physiological P450 chemistry, for example, cyclopropanation and amination reactions. [36,37] These methods have also been extended to improve the properties of this enzyme for biocatalysis including increasing its thermostability and H2O2 peroxygenase activity. [11,19,38]

Certain P450 family members have evolved to naturally function as peroxygenases, such as the H2O2 utilizing CYP152 family of enzymes whose members include CYP152A1 (P450BSβ) [39], CYP152A2 (P450CLA), [40] CYP152B1 (P450SPα), [41] and CYP152L1 (P450 OleT). [42] Recently, inspired by these peroxygenase P450s a single mutation changing threonine 268 to a glutamate was introduced into the P450BM3 heme domain. Threonine 268 is part of the highly conserved acid-alcohol pair in the I-helix used to activate dioxygen. [[5], [6], [7], [8], [9],43] Replacing it with the acidic glutamate residue enabled P450BM3 to hydroxylate tetradecanoic (myristic) acid using hydrogen peroxide as the oxidant (Scheme 1). [44] It has been demonstrated that other reactions such as ethylbenzene hydroxylation and styrene epoxidation could be catalyzed by this P450BM3 heme peroxygenase variant. [45] This method of converting P450 monooxygenases complements others such as the addition of dual-function decoy molecules. [46] The equivalent mutation in other P450 monooxygenases has been used to interrogate the mechanism of the switch in activity to a peroxygenase. [47,48]

Here we demonstrate that the threonine 268 to glutamate heme domain variant (Thr268Glu) can catalyze a range of fatty acid hydroxylation reactions using hydrogen peroxide. The mechanism of fatty acid hydroxylation by this mutant was investigated using [9,9,10,10-d4]-dodecanoic (lauric) acid, which has been selectively deuterated at both the ω-2 and ω-3 positions, to measure an apparent intrinsic kinetic isotope effect. The oxidation of fatty acids was also demonstrated using a flavin based light-activated hydrogen peroxide generating system.

留言 (0)

沒有登入
gif