Unusual cytochrome p450 enzymes and reactions

Abstract

Cytochrome P450 enzymes primarily catalyze mixed-function oxidation reactions, plus some reductions and rearrangements of oxygenated species, e.g. prostaglandins. Most of these reactions can be rationalized in a paradigm involving Compound I, a high-valent iron-oxygen complex (FeO(3+)), to explain seemingly unusual reactions, including ring couplings, ring expansion and contraction, and fusion of substrates. Most P450s interact with flavoenzymes or iron-sulfur proteins to receive electrons from NAD(P)H. In some cases, P450s are fused to protein partners. Other P450s catalyze non-redox isomerization reactions. A number of permutations on the P450 theme reveal the diversity of cytochrome P450 form and function.

Keywords: Cytochrome P450; Enzyme Catalysis; Enzyme Mechanisms; Flavin; Heme; Natural Products; Oxidation-Reduction.

FIGURE 1.

A, major modes of oxidation reactions catalyzed by P450 enzymes. i, Compound I (FeO³⁺) with hydrogen abstraction and oxygen rebound. A variant on this is the initial abstraction of a non-bonded electron from a heteroatom, followed by base-catalyzed rearrangement of the aminium radical (N^+·) to a carbon radical prior to oxygen rebound (2, 3). ii, reaction of an iron peroxy anion (normally an intermediate in the production of FeO³⁺) with an aldehyde, probably the best documented example of this kind of chemistry (4). The process leads to an alkene or aromatic ring, e.g. estrogen synthesis in the aromatase (CYP19A1) reaction (see Fig. 2B). iii, rearrangement of an oxidized entity, exemplified here by the P450 CYP58A1 formation of TXA₂ from PGH₂. B, rearrangements. i, formation and non-enzymatic rearrangement of a carbinolamine and a gem-halohydrin. ii, a stable carbinolamine formed from N-methylcarbazole in P450 oxidations (5, 6).

FIGURE 2.

Possible mechanisms for steroid deformylation reactions catalyzed by the P450 CYP125. A, the important steps involve addition of the ferric-peroxo anion (FeO₂⁻) of CYP125 to the C-26 carbonyl and subsequent radical fragmentation of the peroxyhemiacetal adduct (27). The radical fragmentation of the peroxyhemiacetal adduct leads to formation of an alkene (M1; arrow a) or a one-carbon deficient alcohol (M4; arrow b). The Compound I-catalyzed oxidation of M1 generates a diol (M5) via the acid-catalyzed ring opening of an epoxide intermediate. A C-25 cation may also derive from the single-electron oxidation of the C-25 radical (arrow c). Trapping of the cation by formate or water (arrow d) results in the formation of the C-25 oxyformyl (M2) or the one-carbon reduced alcohol (M4), respectively. Loss of a proton from the C-25 cation may also generate M1 (arrow e). The Compound I-catalyzed oxidation of M4 produces a gem-diol intermediate that dehydrates to a keto compound (M3). B, a possible alternative mechanism involving Baeyer-Villiger oxidation with the ferric-peroxo anion (FeO₂⁻) of CYP125 to yield M2 (see Fig. 1B, ii).

FIGURE 3.

Some unusual P450 reactions. A, proposed mechanism for rearrangement of taxa-4(5),11(12)-diene to 5(12)-oxa-3(11)-cyclotaxane, catalyzed by the P450 CYP725A4 from yew trees (34). B, proposed pathway for nitration of tryptophan (35). C, cyclopropanation by P450_BM-3 (carbene transfer) (36).

FIGURE 4.

Diversity of P450 redox systems and P450 fusion proteins. A selection of distinct types of P450 enzymes and (where relevant) their redox partner systems is shown. The sizes of the boxes are indicative of the lengths of the protein modules. Bound prosthetic groups are indicated in the color-coded domains. A, P450_BM-3 (CYP102A1)-type P450-CPR fusion, also seen for fungal P450_foxy (CYP505)-type systems (54). B, CYP116B-type P450-phthalate dioxygenase reductase fusion (55). C, M. capsulatus P450-FDx fusion CYP51FX (56). D, R. rhodochrous P450-flavodoxin fusion XplA, involved in reductive degradation of explosives (57). E, Pseudomonas fluorescens PfO-1 acyl-CoA dehydrogenase-P450 fusion CYP222A1. This protein is depicted with FAD bound in its N-terminal domain, but there is no report to date of characterization of this protein. F, Mimivirus CYP5253A1, with a P450 fused to a C-terminal domain of uncertain function but containing several potential sites for post-translational modification. G, PpoA dioxygenase/peroxidase-P450 fusion enzyme from A. nidulans, involved in Psi factor production (58). H, P450-hydrolase fusion CYP631B5, involved in mycophenolic acid production (59). I, “stand-alone” P450 that acts without partner proteins, typified by P450_nor (CYP55A)-type nitric-oxide reductase enzymes that interact directly with NAD(P)H, peroxygenase CYP152 P450s that use H₂O₂ to oxidize substrates, P450s that isomerize substrates (e.g. CYP5A1/8A1), and allene oxide synthase (CYP74A) dehydratase P450s. J, typical eukaryotic Class II P450 systems with separate membrane-associated P450 and a CPR partner. K, Class I (mitochondrial) P450 system that interacts with the iron-sulfur protein ADx, which is in turn reduced by ADR. Most bacterial systems use a similar redox apparatus (60). L, variation on system K, in which a flavodoxin replaces the iron-sulfur protein. This type of system supports CYP176A1 (P450_cin); enables Citrobacter braakii to catabolize cineole; and can also reduce CYP107H1 (P450_BioI), involved in B. subtilis biotin synthesis (61, 62). M, heme-free EryCII P450-like protein devoid of a cysteine proximal ligand. EryCII is an allosteric activator of the glycosyltransferase EryCIII in the production of erythromycin D in S. erythraea (63).