A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs

Structural interactions that enable electron transfer to cytochrome‐P450 (CYP450) from its redox partner CYP450‐reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane‐bound functional complex to reveal interactions between the full‐length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome‐b₅ (cyt‐b₅), Arg 125 on the C‐helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein–protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.     

Significance of Protein–Substrate Hydrogen Bonding for the Selectivity of P450-Catalysed Oxidations

P450_cin and P450_cam are bacterial cytochromes P450 that specifically hydroxylate bicyclic monoterpenes. Protein–substrate H bonding has been previously proposed as crucial in the selectivity of P450_cin oxidations, but not as essential for P450_cam. To examine the difference in importance of H bonds in these two model P450s, the P450-catalysed oxidation products from thiocamphor were compared. Surprisingly, both P450s oxidised thiocamphor predominantly to the corresponding S-oxides, in contrast to previous reports, and this is the first report of P450-catalysed sulfine generation from a thioketone. Additionally, the result emphasised the importance of the protein–substrate H bond to selectivity in both P450_cin and P450_cam. The H bonding in P450_cam was re-examined using camphane, another substrate for which the protein–substrate H bond is absent. The results indicated that both H bonding and hydrophobic interactions between substrate and protein play a role in selectivity. Interestingly, the protein–substrate H bond was not a factor in substrate affinity for the enzyme.     

细胞色素P-450模拟酶的合成及其用于常温常压下催化氧化苯为苯酚的研究

本文合成了三种具有不同取代基的铁卟啉配合物,其中氯化四—(4—羟基—3—甲氧苯基)卟啉铁(Ⅲ)是迄今未见报道的新化合物。用它们分别与硫醇化合物和氧一起,构成了新型的细胞色素P—450的模拟体系。实验表明,这些体系具有显著的吸氧活性并能在常温常压下将苯一步氧化为苯酚。文中还对结构与性能之间的关系进行了探讨。在实验的基础上,对羟化机制提出了初步的设想。     

Engineering P450 Peroxygenase to Catalyze Highly Enantioselective Epoxidation of cis‐β‐Methylstyrenes

P450 119 peroxygenase and its site‐directed mutants are discovered to catalyze the enantioselective epoxidation of methyl‐substituted styrenes. Two new site‐directed P450 119 mutants, namely T213Y and T213M, which were designed to improve the enantioselectivity and activity for the epoxidation of styrene and its methyl substituted derivatives, were studied. The T213M mutant is found to be the first engineered P450 peroxygenase that shows highly enantioselective epoxidation of cis‐β‐methylstyrenes, with up to 91 % ee. Molecular modeling studies provide insights into the different catalytic activity of the T213M mutant and the T213Y mutant in the epoxidation of cis‐β‐methylstyrene. The results of the calculations also contribute to a better understanding of the substrate specificity and configuration control for the regio‐ and stereoselective peroxygenation catalyzed by the T213M mutant.     

Metabolic‐intermediate complex formation with cytochrome P450: Theoretical studies in elucidating the reaction pathway for the generation of reactive nitroso intermediate

Mechanism‐based inhibition (MBI) of cytochrome P450 (CYP) can lead to drug–drug interactions and often to toxicity. Some aliphatic and aromatic amines can undergo biotransformation reactions to form reactive metabolites such as nitrosoalkanes, leading to MBI of CYPs. It has been proposed that the nitrosoalkanes coordinate with the heme iron, forming metabolic‐intermediate complex (MIC), resulting in the quasi‐irreversible inhibition of CYPs. Limited mechanistic details regarding the formation of reactive nitroso intermediate and its coordination with heme‐iron have been reported. A quantum chemical analysis was performed to elucidate potential reaction pathways for the generation of nitroso intermediate and the formation of MIC. Elucidation of the energy profile along the reaction path, identification of three‐dimensional structures of reactive intermediates and transition states, as well as charge and spin density analyses, were performed using the density functional B3LYP method. The study was performed using Cpd I [iron (IV‐oxo] heme porphine with SH^? as the axial ligand) to represent the catalytic domain of CYP, simulating the biotransformation process. Three pathways: (i) N‐oxidation followed by proton shuttle, (ii) N‐oxidation followed by 1,2‐H shift, and (iii) H‐abstraction followed by rebound mechanism, were studied. It was observed that the proton shuttle pathway was more favorable over the whole reaction leading to reactive nitroso intermediate. This study revealed that the MIC formation from a primary amine is a favorable exothermic process, involving eight different steps and preferably takes place on the doublet spin surface of Cpd I . The rate‐determining step was identified to be the first N‐oxidation of primary amine. © 2012 Wiley Periodicals, Inc.     

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.     

Biomimetic oxidation of N-nitrosodibenzylamine with molecular oxygen catalysed by chemical cytochrome P-450 in AOT reverse micelles

Moderate yields of benzaldehyde, benzyl alcohol and benzylamine are obtained by the biomimetic oxidation of N-nitrosodibenzylamine with molecular oxygen catalysed by water soluble anionic manganese(III) 5,10,15,20-tetraphenylporphyrin acetate/sodium dithionite/methylene blue in aerosol-OT (AOT) reverse micelles, under phase transfer conditions with AOT concentration higher than 10⁻³M. The formation of α-hydroxy-N-nitrosodibenzylamine and its decomposition products, benzaldehyde and benzyl alcohol in reverse micellar systems are governed by the ratio of water and AOT, pH and other changes in the microenvirpnment.     

Innovational combination of hetero-bifunctional N-PEG quinoline scaffolds derivatives with improved anticancer activity against breast and colon cancer cell lines and P-glycoprotein,cytochrome p450 enzyme activity prediction

Polyethylene glycol (PEG) is a polymer that is widely used as a carrier for drug delivery systems (DDS). A library of N-PEGylated quinoline derivatives of PEG molecular weight 200 was prepared rapidly after the activation of PEGs using maleic anhydride. Quinoline with a polymer backbone is essential as new material. PEG is a water-soluble nonionic polymer approved by food and drug organizations for medicine applications. Because of its nontoxic grapheme, it is widely utilized in numerous biochemical, cosmetic, pharmaceutical, and industrialized applications. The modern SwissADME is a web tool that stretches free admittance to a pool of hasty, yet solid, clarifying models for physicochemical properties, pharmacokinetics, and therapeutic science. The present facile synthetic strategy can be a practical approach for incorporating polymeric carriers conjugated with drug moieties, either in the backbone of the polymer or as a terminal and pendant group on the polymer chains.     

Convergent asymmetric synthesis of (+)-aureothin employing an oxygenase-mediated resolution step

Need an enzymatic push? The desymmetrization of α,α'-dimethoxy-γ-pyrone allows the convergent and rapid preparation of the complete carbon skeleton of (+)-aureothin. The final step in the synthesis of the target molecule is the regiodivergent parallel kinetic resolution promoted by cytochrome P450 monooxygenase AurH to deliver the enantiopure natural product.     

Structural and Dynamic Basis of Human Cytochrome P450 7B1: A Survey of Substrate Selectivity and Major Active Site Access Channels

Cytochrome P450 (CYP) 7B1 is a steroid cytochrome P450 7α‐hydroxylase that has been linked directly with bile salt synthesis and hereditary spastic paraplegia type 5 (SPG5). The enzyme provides the primary metabolic route for neurosteroids dehydroepiandrosterone (DHEA), cholesterol derivatives 25‐hydroxycholesterol (25‐HOChol), and other steroids such as 5α‐androstane‐3β,17β‐diol (anediol), and 5α‐androstene‐3β,17β‐diol (enediol). A series of investigations including homology modeling, molecular dynamics (MD), and automatic docking, combined with the results of previous experimental site‐directed mutagenesis studies and access channels analysis, have identified the structural features relevant to the substrate selectivity of CYP7B1. The results clearly identify the dominant access channels and critical residues responsible for ligand binding. Both binding free energy analysis and total interaction energy analysis are consistent with the experimental conclusion that 25‐HOChol is the best substrate. According to 20 ns MD simulations, the Phe cluster residues that lie above the active site, particularly Phe489, are proposed to merge the active site with the adjacent channel to the surface and accommodate substrate binding in a reasonable orientation. The investigation of CYP7B1–substrate binding modes provides detailed insights into the poorly understood structural features of human CYP7B1 at the atomic level, and will be valuable information for drug development and protein engineering.     

Second-Coordination Sphere Effects on Selectivity and Specificity of Heme and Nonheme Iron Enzymes

Mononuclear iron-containing enzymes are highly versatile oxidants that often react stereospecifically and/or regioselectively with substrates. Combined experimental and computational studies on heme monooxygenases, nonheme iron dioxygenases and halogenases have revealed the intricate details of the second-coordination sphere, which determine this specificity and selectivity. These second-coordination sphere effects originate from the positioning of the substrate and oxidant, which involve the binding of the co-factors and substrate into the active site of the protein. In addition, some enzymes affect the selectivity and reactivity through charge-stabilization from nearby bound cations/anions, an induced electric field or through the positioning of salt bridges and hydrogen-bonding interactions to first-coordination sphere iron ligands and/or the substrate. Examples of all of these second-coordination sphere effects in iron-containing enzymes and how these influence structure and reactivity are given.