Enabling Aromatic Hydroxylation in a Cytochrome P450 Monooxygenase Enzyme through Protein Engineering

The cytochrome P450 (CYP) family of heme monooxygenases catalyse the selective oxidation of C−H bonds under ambient conditions. The CYP199A4 enzyme from Rhodopseudomonas palustris catalyses aliphatic oxidation of 4-cyclohexylbenzoic acid but not the aromatic oxidation of 4-phenylbenzoic acid, due to the distinct mechanisms of aliphatic and aromatic oxidation. The aromatic substrates 4-benzyl-, 4-phenoxy- and 4-benzoyl-benzoic acid and methoxy-substituted phenylbenzoic acids were assessed to see if they could achieve an orientation more amenable to aromatic oxidation. CYP199A4 could catalyse the efficient benzylic oxidation of 4-benzylbenzoic acid. The methoxy-substituted phenylbenzoic acids were oxidatively demethylated with low activity. However, no aromatic oxidation was observed with any of these substrates. Crystal structures of CYP199A4 with 4-(3′-methoxyphenyl)benzoic acid demonstrated that the substrate binding mode was like that of 4-phenylbenzoic acid. 4-Phenoxy- and 4-benzoyl-benzoic acid bound with the ether or ketone oxygen atom hydrogen-bonded to the heme aqua ligand. We also investigated whether the substitution of phenylalanine residues in the active site could permit aromatic hydroxylation. Mutagenesis of the F298 residue to a valine did not significantly alter the substrate binding position or enable the aromatic oxidation of 4-phenylbenzoic acid; however the F182L mutant was able to catalyse 4-phenylbenzoic acid oxidation generating 2′-hydroxy-, 3′-hydroxy- and 4′-hydroxy metabolites in a 83 : 9 : 8 ratio, respectively. Molecular dynamics simulations, in which the distance and angle of attack were considered, demonstrated that in the F182L variant, in contrast to the wild-type enzyme, the phenyl ring of 4-phenylbenzoic acid attained a productive geometry for aromatic oxidation to occur.     

The crystal structures of 4-methoxybenzoate bound CYP199A2 and CYP199A4: structural changes on substrate binding and the identification of an anion binding site

The crystal structures of the 4-methoxybenzoate bound forms of cytochrome P450 enzymes CYP199A2 and CYP199A4 from the Rhodopseudomonas palustris strains CGA009 and HaA2 have been solved. The structures of these two enzymes, which share 86% sequence identity, are very similar though some differences are found on the proximal surface. In these structures the enzymes have a closed conformation, in contrast to the substrate-free form of CYP199A2 where an obvious substrate access channel is observed. The switch from an open to a closed conformation arises from pronounced residue side-chain movements and alterations of ion pair and hydrogen bonding interactions at the entrance of the access channel. A chloride ion bound just inside the protein surface caps the entrance to the active site and protects the substrate and the heme from the external solvent. In both structures the substrate is held in place via hydrophobic and hydrogen bond interactions. The methoxy group is located over the heme iron, accounting for the high activity and selectivity of these enzymes for oxidative demethylation of the substrate. Mutagenesis studies on CYP199A4 highlight the involvement of hydrophobic (Phe185) and hydrophilic (Arg92, Ser95 and Arg243) amino acid residues in the binding of para-substituted benzoates by these enzymes.     

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.     

Regioselective Hydroxylation of C12–C15 Fatty Acids with Fluorinated Substituents by Cytochrome P450 BM3

We demonstrate herein that wild‐type cytochrome P450 BM3 can recognize non‐natural substrates, such as fluorinated C₁₂–C₁₅ chain‐length fatty acids, and show better catalysis for their efficient conversion. Although the binding affinities for fluorinated substrates in the P450 BM3 pocket are marginally lower than those for non‐fluorinated substrates, spin‐shift measurements suggest that fluoro substituents at the ω‐position can facilitate rearrangement of the dynamic structure of the bulk‐water network within the hydrophobic pocket through a micro desolvation process to expel the water ligand of the heme iron that is present in the resting state. A lowering of the Michaelis–Menten constant (K_m), however, indicates that fluorinated fatty acids are indeed better substrates compared with their non‐fluorinated counterparts. An enhancement of the turnover frequencies (k_cat) for electron transfer from NADPH to the heme iron and for C? H bond oxidation by compound I (Cpd I) to yield the product suggests that the activation energies associated with going from the enzyme–substrate (ES state) to the corresponding transition state (ES^≠ state) are significantly lowered for both steps in the case of the fluorinated substrates. Delicate control of the regioselectivity by the fluorinated terminal methyl groups of the C₁₂–C₁₅ fatty acids has been noted. Despite the fact that residues Arg47/Tyr51/Ser72 exert significant control over the hydroxylation of the subterminal carbon atoms toward the hydrocarbon tail, the fluorine substituent(s) at the ω‐position affects the regioselective hydroxylation. For substrate hydroxylation, we have found that fluorinated lauric acids probably give a better structural fit for the heme pocket than fluorinated pentadecanoic acid, even though pentadecanoic acid is by far the best substrate among the reported fatty acids. Interestingly, 12‐fluorododecanoic acid, with only one fluorine atom at the terminal methyl group, exhibits a comparable turnover frequency to that of pentadecanoic acid. Thus, fluorination of the terminal methyl group introduces additional interactions of the substrate within the hydrophobic pocket, which influence the electron transfers for both dioxygen activation and the controlled oxidation of aliphatics mediated by high‐valent oxoferryl species.     

Structural and dynamical basis of broad substrate specificity, catalytic mechanism, and inhibition of cytochrome P450 3A4

Cytochrome P450 (CYP) 3A4 is responsible for the oxidative degradation of more than 50% of clinically used drugs. By means of molecular dynamics simulations with the newly developed force field parameters for the heme-thiolate group and its dioxygen adduct, we examine the differences in structural and dynamic properties between CYP3A4 in the resting form and its complexes with the substrate progesterone and the inhibitor metyrapone. The results indicate that the broad substrate specificity of CYP3A4 stems from the malleability of a loop (residues 211-218) that resides in the vicinity of the channel connecting the active site and bulk solvent. However, the high-amplitude motion of the flexible loop is found to be damped out upon binding of the inhibitor or the substrate in the active site. In the resting form of CYP3A4, a structural water molecule is bound to the sixth coordination position of the heme iron, stabilizing the octahedral coordination geometry. In addition to the direct coordination of metyrapone to the heme iron, the hydrogen bond interaction between the inhibitor carbonyl group and the side chain of Ser119 also contributes significantly to stabilizing the CYP3A4-metyrapone complex. On the other hand, progesterone is stabilized in the active site by the formation of two hydrogen bonds with Ser119 and Arg106, as well as by the van der Waals interactions with the heme and hydrophobic residues. The structural and dynamic features of the CYP3A4-progesterone complex indicate that the oxidative degradation of progesterone occurs through hydroxylation at the C16 position by the reactive oxygen coordinated to the heme iron.     

Insights into the Desaturation of Cyclopeptin and its C3 Epimer Catalyzed by a non‐Heme Iron Enzyme: Structural Characterization and Mechanism Elucidation

AsqJ, an iron(II)‐ and 2‐oxoglutarate‐dependent enzyme found in viridicatin‐type alkaloid biosynthetic pathways, catalyzes sequential desaturation and epoxidation to produce cyclopenins. Crystal structures of AsqJ bound to cyclopeptin and its C3 epimer are reported. Meanwhile, a detailed mechanistic study was carried out to decipher the desaturation mechanism. These findings suggest that a pathway involving hydrogen atom abstraction at the C10 position of the substrate by a short‐lived Fe^IV‐oxo species and the subsequent formation of a carbocation or a hydroxylated intermediate is preferred during AsqJ‐catalyzed desaturation.     

Defining CYP3A4 structural responses to substrate binding. Raman spectroscopic studies of a nanodisc-incorporated mammalian cytochrome P450

Resonance Raman (RR) spectroscopy is used to help define active site structural responses of nanodisc-incorporated CYP3A4 to the binding of three substrates: bromocriptine (BC), erythromycin (ERY), and testosterone (TST). We demonstrate that nanodisc-incorporated assemblies reveal much more well-defined active site RR spectroscopic responses as compared to those normally obtained with the conventional, detergent-stabilized, sampling strategies. While ERY and BC are known to bind to CYP3A4 with a 1:1 stoichiometry, only the BC induces a substantial conversion from low- to high-spin state, as clearly manifested in the RR spectra acquired herein. The third substrate, TST, displays significant homotropic interactions within CYP3A4, the active site binding up to 3 molecules of this substrate, with the functional properties varying in response to binding of individual substrate molecules. While such behavior seemingly suggests the possibility that each substrate binding event induces functionally important heme structural changes, up to this time spectroscopic evidence for such structural changes has not been available. The current RR spectroscopic studies show clearly that accommodation of different size substrates, and different loading of TST, do not significantly affect the structure of the substrate-bound ferric heme. However, it is here demonstrated that the nature and number of bound substrates do have an extraordinary influence on the conformation of bound exogenous ligands, such as CO or dioxygen and its reduced forms, implying an effective mechanism whereby substrate structure can impact reactivity of intermediates so as to influence function, as reflected in the diverse reactivity of this drug metabolizing cytochrome.     

N‐Heterocyclic Carbene Iron(III) Porphyrin‐Catalyzed Intramolecular C(sp3)–H Amination of Alkyl Azides

Metal‐catalyzed intramolecular C?H amination of alkyl azides constitutes an appealing approach to alicyclic amines; challenges remain in broadening substrate scope, enhancing regioselectivity, and applying the method to natural product synthesis. Herein we report an iron(III) porphyrin bearing axial N‐heterocyclic carbene ligands which catalyzes the intramolecular C(sp³)–H amination of a wide variety of alkyl azides under microwave‐assisted and thermal conditions, resulting in selective amination of tertiary, benzylic, allylic, secondary, and primary C?H bonds with up to 95 % yield. 14 out of 17 substrates were cyclized selectively at C4 to give pyrrolidines. The regioselectivity at C4 or C5 could be tuned by modifying the reactivity of the C5–H bond. Mechanistic studies revealed a concerted or a fast re‐bound mechanism for the amination reaction. The reaction has been applied to the syntheses of tropane, nicotine, cis‐octahydroindole, and leelamine derivatives.     

Evolved Aliphatic Halogenases Enable Regiocomplementary C−H Functionalization of a Pharmaceutically Relevant Compound

Non‐heme iron halogenases are synthetically valuable biocatalysts that are capable of halogenating unactivated sp³‐hybridized carbon centers with high stereo‐ and regioselectivity. The reported substrate scope of these enzymes, however, is limited primarily to the natural substrates and their analogues. We engineered the halogenase WelO5* for chlorination of a martinelline‐derived fragment. Using structure‐guided evolution, a halogenase variant with a more than 290‐fold higher total turnover number and a 400‐fold higher apparent k_cat compared to the wildtype enzyme was generated. Moreover, we identified key positions in the active site that allow direction of the halogen to different positions in the target substrate. This is the first example of enzyme engineering to expand the substrate scope of a non‐heme iron halogenase beyond the native indole‐alkaloid‐type substrates. The highly evolvable nature of WelO5* underscores the usefulness of this enzyme family for late‐stage halogenation.     

Mimicking the Aromatic‐Ring‐Cleavage Activity of Gentisate‐1,2‐Dioxygenase by a Nonheme Iron Complex

Gentisate‐1,2‐dioxygenase (GDO), a nonheme iron enzyme in the cupin superfamily, catalyzes the cleavage of the aromatic‐ring of 2,5‐dihydroxybenzoic acid (gentisic acid) to form maleylpyruvic acid in the microbial aerobic degradation of aromatic compounds. To develop a functional model of GDO, we have isolated a nonheme iron(II) complex, [(Tp^Ph2)Fe^II(DHN‐H)] (Tp^Ph2=hydrotris(3,5‐diphenylpyrazole‐1‐yl)borate, DHN‐H=1,4‐dihydroxy‐2‐naphthoate). In the reaction with O₂, the biomimetic complex oxidatively cleaves the aromatic ring of the coordinated substrate with the incorporation of both the oxygen atoms from molecular oxygen into the cleavage product. The presence of para‐hydroxy group on the substrate plays a crucial role in directing the aromatic‐ring cleaving reaction.     

Pyrene.pyrene complexes at the active site of cytochrome P450 3A4: evidence for a multiple substrate binding site

Cytochrome P450 monooxygenases (CYPs) metabolize nearly all drugs and toxins. Recently, it has become clear that CYPs exhibit both homotropic and heterotropic allosteric kinetics for many substrates. However, the mechanism of cooperative kinetics has not been established for any specific human CYP/substrate combination. Suggested mechanisms include binding of multiple substrates within distinct, static, subsites of a single large active site or binding of multiple substrates within a single fluid active site. CYP3A4 hydroxylates pyrene with positive cooperativity. Therefore, experiments were designed to exploit the fluorescence properties of pyrene, which diagnostically distinguish between pyrene.pyrene complexes versus spatially separated pyrene substrates. Pyrene complexes (excimers) yield an emission spectrum clearly distinct from pyrene monomers. In lipid-free aqueous/glycerol solutions of CYP3A4, addition of pyrene affords a concentration-dependent low-spin to high-spin conversion of the CYP3A4 heme prosthetic group, indicating occupancy of the active site by pyrene. Under the same conditions, in the presence of CYP3A4 but not other heme proteins, the excimer/monomer ratio (E/M) of pyrene was decreased in emission spectra, compared to pyrene alone. However, excitation spectra indicate a CYP3A4-dependent increase in the wavelength shift for the excimer excitation spectrum versus the monomer excitation spectrum, as well as changes in the excimer excitation peak shape and vibronic structure. These changes are reversed by the CYP3A4 substrate testosterone. Together, the results demonstrate that pyrene.pyrene ground-state complexes occupy the CYP3A4 active site, and they provide the first spectroscopic evidence for substrate complexes within a single fluid active site. Functional implications include the possibility that turnover rate, regioselectivity, and stereoselectivity of the reaction are determined by the substrate.substrate complex rather than individual substrates.     

Sulfonamide‐Promoted Palladium(II)‐Catalyzed Alkylation of Unactivated Methylene C(sp3)H Bonds with Alkyl Iodides

The alkylation of unactivated β‐methylene C(sp³)? H bonds of α‐amino acid substrates with a broad range of alkyl iodides using Pd(OAc)₂ as the catalyst is described. The addition of NaOCN and 4‐Cl‐C₆H₄SO₂NH₂ was found to be crucial for the success of this transformation. The reaction is compatible with a diverse array of functional groups and proceeds with high diastereoselectivity. Furthermore, various β,β‐hetero‐dialkyl‐ and β‐alkyl‐β‐aryl‐α‐amino acids were prepared by sequential C(sp³)? H functionalization of an alanine‐derived substrate, thus providing a versatile strategy for the stereoselective synthesis of unnatural β‐disubstituted α‐amino acids.     

Sulfonamide‐Promoted Palladium(II)‐Catalyzed Alkylation of Unactivated Methylene C(sp3)?H Bonds with Alkyl Iodides

The alkylation of unactivated β‐methylene C(sp³) H bonds of α‐amino acid substrates with a broad range of alkyl iodides using Pd(OAc)₂ as the catalyst is described. The addition of NaOCN and 4‐Cl‐C₆H₄SO₂NH₂ was found to be crucial for the success of this transformation. The reaction is compatible with a diverse array of functional groups and proceeds with high diastereoselectivity. Furthermore, various β,β‐hetero‐dialkyl‐ and β‐alkyl‐β‐aryl‐α‐amino acids were prepared by sequential C(sp³) H functionalization of an alanine‐derived substrate, thus providing a versatile strategy for the stereoselective synthesis of unnatural β‐disubstituted α‐amino acids.     

Enzyme Multifunctionality by Control of Substrate Positioning Within the Catalytic Cycle—A QM/MM Study of Clavaminic Acid Synthase

Clavaminic acid synthase from Streptomyces clavuligerus is an Fe^II/2-oxoglutarate-dependent dioxygenase, crucial for the biosynthesis of the β-lactamase inhibitor clavulanic acid. It catalyses three consecutive oxidative reactions, that is, hydroxylation, cyclisation and desaturation, in a single binding cavity. As follows from the results of this QM/MM study, CAS versatility and selectivity depends on the binding cavity, which interplays differently with the substrate for each reaction. The enzyme–substrate interactions affect the substrate's ability to re-position during the reaction, either constraining it in its primary position, which impedes processes other than oxygen rebound, or allowing change, which facilitates desaturation. This differential effect originates from two aspartate residues, which strongly interact with the guanidine group of the hydroxylation substrate and stabilise the orientation of the molecule. These residues interact less effectively with the smaller amine group of the desaturation substrate(s), aiding their re-positioning and the subsequent formation of a double bond.     

Synergistic Substrate and Oxygen Activation in Salicylate Dioxygenase Revealed by QM/MM Simulations

Salicylate 1,2‐dioxygenase (SDO) is the first enzyme to be discovered to catalyze the oxidative cleavage of a monohydroxylated aromatic compound, namely salicylate, instead of the well‐known electron‐rich substrates. We have investigated the mechanism of dioxygen activation in SDO by QM/MM calculations. Our study reveals that the non‐heme Fe^II center in SDO activates salicylate and O₂ synergistically through a strong covalent interaction to facilitate the reductive cleavage of O₂. A covalent salicylate–Fe^II–O₂ complex is the reactive oxygen species in this case, and its electronic structure is best described as being between the two limiting cases, Fe^II?O₂ and Fe^II?O₂^.?, with partial electron transfer from the activated salicylate to O₂ via the Fe center. Thus SDO employs a synergistic strategy of substrate and oxygen activation to carry out the catalytic reaction, which is unprecedented in the family of iron dioxygenases. Moreover, O₂ activation in SDO happens without the assistance of a proton source. Our study essentially shows a new mechanistic possibility for O₂ activation.     

The Cobalt Way to Angucyclinones: Asymmetric Total Synthesis of the Antibiotics (+)‐Rubiginone B2, (−)‐Tetrangomycin,and (−)‐8‐O‐Methyltetrangomycin

A cobalt(I)‐mediated convergent and asymmetric total synthesis of angucyclinones with an aromatic B ring has been developed. In the course of our research, we synthesized three naturally occurring anguclinone derivatives, namely, (+)‐rubiginone B₂ ( 1 ), (?)‐8‐O‐methyltetrangomycin ( 2 ), and (?)‐tetrangomycin ( 3 ). By combining 3‐hydroxybenzoic acid, 3‐methoxybenzoic acid, citronellal, and geraniol as starting materials in a convergent way, we were able to synthesize chiral triyne chains, which were cyclized with [CpCo(C₂H₄)₂] (Cp=cyclopentadienyl) by means of an intramolecular [2+2+2] cycloaddition to their corresponding tetrahydrobenzo[a]anthracenes. Successive oxidation and deprotection steps led to the above‐mentioned natural products 1 – 3 .     

Synthesis and Characterization of New 3‐(3‐Hydroxyphenyl)‐4‐ alkyl‐3,4‐dihydrobenzo[e][1,3]oxazepine‐1,5‐dione Compounds

A series of 3‐(3‐hydroxyphenyl)‐4‐alkyl‐3,4‐dihydrobenzo[e][1,3]oxazepine‐1,5‐dione compounds with general formula C_nH_2n+1CNO(CO)₂C₆H₄(C₆H₄OH) in which n are even parity numbers from 2 to 18. The structure determinations on these compounds were performed by FT‐IR spectroscopy which indicated that the terminal alkyl chain attached to the oxazepine ring was fully extended. Conformational analysis in DMSO at ambient temperature was carried out for the first time via high resolution ¹H NMR and ¹³C NMR spectroscopy.     

Differences and Comparisons of the Properties and Reactivities of Iron(III)–hydroperoxo Complexes with Saturated Coordination Sphere

Heme and nonheme monoxygenases and dioxygenases catalyze important oxygen atom transfer reactions to substrates in the body. It is now well established that the cytochrome P450 enzymes react through the formation of a high‐valent iron(IV)–oxo heme cation radical. Its precursor in the catalytic cycle, the iron(III)–hydroperoxo complex, was tested for catalytic activity and found to be a sluggish oxidant of hydroxylation, epoxidation and sulfoxidation reactions. In a recent twist of events, evidence has emerged of several nonheme iron(III)–hydroperoxo complexes that appear to react with substrates via oxygen atom transfer processes. Although it was not clear from these studies whether the iron(III)–hydroperoxo reacted directly with substrates or that an initial O?O bond cleavage preceded the reaction. Clearly, the catalytic activity of heme and nonheme iron(III)–hydroperoxo complexes is substantially different, but the origins of this are still poorly understood and warrant a detailed analysis. In this work, an extensive computational analysis of aromatic hydroxylation by biomimetic nonheme and heme iron systems is presented, starting from an iron(III)–hydroperoxo complex with pentadentate ligand system (L₅²). Direct C?O bond formation by an iron(III)–hydroperoxo complex is investigated, as well as the initial heterolytic and homolytic bond cleavage of the hydroperoxo group. The calculations show that [(L₅²)Fe^III(OOH)]²⁺ should be able to initiate an aromatic hydroxylation process, although a low‐energy homolytic cleavage pathway is only slightly higher in energy. A detailed valence bond and thermochemical analysis rationalizes the differences in chemical reactivity of heme and nonheme iron(III)–hydroperoxo and show that the main reason for this particular nonheme complex to be reactive comes from the fact that they homolytically split the O?O bond, whereas a heterolytic O?O bond breaking in heme iron(III)–hydroperoxo is found.     

Selenolate complexes of CYP101 and the heme-bound hHO-1/H25A proximal cavity mutant

Thiolate and selenolate complexes of CYP101 (P450cam) and the H25A proximal cavity mutant of heme-bound human heme oxygenase-1 (hHO-1) have been examined by UV-vis spectroscopy. Both thiolate and selenolate ligands bound to the heme distal side in CYP101 and gave rise to characteristic hyperporphyrin spectra. Thiolate ligands also bound to the proximal side of the heme in the cavity created by the H25A mutation in hHO-1, giving a Soret absorption similar to that of the H25C hHO-1 mutant. Selenolate ligands also bound to this cavity mutant under anaerobic conditions but reduced the heme iron to the ferrous state, as shown by the formation of a ferrous CO complex. Under aerobic conditions, the selenolate ligand but not the thiolate ligand was rapidly oxidized. These results indicate that selenocysteine-coordinated heme proteins will not be stable species in the absence of a redox potential stabilizing effect.     

Redox Self‐Adaptation of a Nitrene Transfer Catalyst to the Substrate Needs

The development of iron catalysts for carbon–heteroatom bond formation, which has attracted strong interest in the context of green chemistry and nitrene transfer, has emerged as the most promising way to versatile amine synthetic processes. A diiron system was previously developed that proved efficient in catalytic sulfimidations and aziridinations thanks to an Fe^IIIFe^IV active species. To deal with more demanding benzylic and aliphatic substrates, the catalyst was found to activate itself to a Fe^IIIFe^IVL^. active species able to catalyze aliphatic amination. Extensive DFT calculations show that this activation event drastically enhances the electron affinity of the active species to match the substrates requirements. Overall this process consists in a redox self‐adaptation of the catalyst to the substrate needs.