Structures Of P450s Research Articles

Diverse reactions catalyzed by cytochrome P450 and biosynthesis of steroid hormone.

Steroid hormones modulate numerous physiological processes in various higher organisms. Research on the physiology, biosynthesis, and metabolic degradation of steroid hormones is crucial for developing drugs, agrochemicals, and anthelmintics. Most steroid hormone biosynthetic pathways, excluding those in insects, have been elucidated, and the roles of several cytochrome P450s (CYPs, P450s), heme (iron protoporphyrin IX)-containing monooxygenases, have been identified. Specifically, P450s of the animal steroid hormone biosynthetic pathways and their three dimensional structures and reaction mechanisms have been extensively studied; however, the mechanisms of several uncommon P450 reactions involved in animal steroid hormone biosynthesis and structures and reaction mechanisms of various P450s involved in plant and insect steroid hormone biosynthesis remain unclear. Recently, we determined the crystal structure of P450 responsible for the first and rate-determining step in brassinosteroids biosynthesis and clarified the regio- and stereo-selectivity in the hydroxylation reaction mechanism. In this review, we have outlined the general catalytic cycle, reaction mechanism, and structure of P450s. Additionally, we have described the recent advances in research on the reaction mechanisms of steroid hormone biosynthesis-related P450s, some of which catalyze unusual P450 reactions including C–C bond cleavage reactions by utilizing either a heme–peroxo anion species or compound I as an active oxidizing species. This review article is an extended version of the Japanese article, Structure and mechanism of cytochrome P450s involved in steroid hormone biosynthesis, published in SEIBUTSU BUTSURI Vol. 61, p. 189–191 (2021).

Mechanisms of Cytochrome P450-Catalyzed Oxidations.

Enzymes are complex biological catalysts and are critical to life. Most oxidations of chemicals are catalyzed by cytochrome P450 (P450, CYP) enzymes, which generally utilize mixed-function oxidase stoichiometry, utilizing pyridine nucleotides as electron donors: NAD(P)H + O2 + R → NAD(P)+ + RO + H2O (where R is a carbon substrate and RO is an oxidized product). The catalysis of oxidations is largely understood in the context of the heme iron-oxygen complex generally referred to as Compound I, formally FeO3+, whose basis was in peroxidase chemistry. Many X-ray crystal structures of P450s are now available (≥ 822 structures from ≥146 different P450s) and have helped in understanding catalytic specificity. In addition to hydroxylations, P450s catalyze more complex oxidations, including C-C bond formation and cleavage. Enzymes derived from P450s by directed evolution can even catalyze more unusual reactions, e.g. cyclopropanation. Current P450 questions under investigation include the potential role of the intermediate Compound 0 (formally FeIII-O2 -) in catalysis of some reactions, the roles of high- and low-spin forms of Compound I, the mechanism of desaturation, the roles of open and closed structures of P450s in catalysis, the extent of processivity in multi-step oxidations, and the role of the accessory protein cytochrome b 5. More global questions include exactly how structure drives function, prediction of catalysis, and roles of multiple protein conformations.

Comparative Analysis of Full-Length Cytochromes P450 in Complexes with Cytochrome B5 in Membrane

Molecular models of complexes of full-length cytochrome P450s with cytochrome B5 (CYB5) were developed based on experimental structures of CYB5 (2m33) and several P450s in closed (2q6n, 1tqn, 3e4e, 3eqm, 3ruk) and open (2bdm) conformations and structural constraints derived from mutagenesis, mass-spectrometry, and NMR studies. Models of transmembrane parts of the complexes formed by two antiparallel α-helices were generated using original energy functions for quantification of helix interactions in membranes. Our PPM method was used to assess the spatial orientations of complexes in membrane. We found that CYB5 (helices 41-43, 48-54, and 60-66) binds to the proximal groove of P450s formed by helices B, C, J', K, L, the C-D loop, and the K''-L meander region. In all models, acidic residues E61 and D65 of CYB5 form ionic pairs with basic residues of P450 (from helices B, C, and the K''-L meander), whereas propionate groups of the heme interact with conserved arginine from helix C (R133 in CYPB4). However, CYB5 orientations vary in complexes with different P450s and depend on the conformational state of P450s. In particular, CYB5 interacts similarly with closed forms of CYP2E1, CYP17A1, and CYP2C19: E58 of CYB5 forms a contact with helix B of P450 and E42/E48 of CYB5 interact with helices J', K, and L of P450. A partly different binding mode was identified for complexes of CYP3A4 and CYP2B4, where E42/E48 of CYB5 contact with the K''-L meander and T70/D71 of CYB5 interact with the C-D loop. Comparison of complexes with CYP2B4 in closed and open conformations shows that open conformation has larger protein-protein contact surface, shorter heme-to-heme distance, deeper penetration of the complex into membrane, and provides additional interprotein hydrophobic interactions (V50-I435 and V66-L437) which stabilize the complex.

Structure and Function of Cytochrome P450S in Insect Adaptation to Natural and Synthetic Toxins: Insights Gained from Molecular Modeling

Over evolutionary time, insect herbivores have adapted to the presence of natural toxins and more recently to synthetic insecticides in or on the plants they consume. Biochemical analyses and molecular modeling of the cytochrome P450 monooxygenases (P450s) that metabolize these compounds have provided insight into the many variations affecting their catalytic activity. Phylogenetically distinct P450s may metabolize similar substrates, and phylogenetically similar P450s may metabolize different substrates; as well, some P450s process broad arrays of both phytochemicals and synthetic insecticides, while closely related P450s are restricted to a narrow range of phytochemicals. Mapped on the predicted three-dimensional structures of insect P450s developed from available mammalian P450 crystal structures, differences in multiple regions of the insect proteins reveal the evolutionary processes occurring as P450 genes have duplicated and diverged. Analyses of site-directed mutants in select lepidopteran and dipteran P450s demonstrate that slight changes in the catalytic site, the putative product release channel, and the proximal surface (interacting with electron transfer partners such as cytochrome P450 reductase and cytochrome b5) yield pronounced activity differences. Additionally, changes in the catalytic site and in the linker region preceding the proline-hinge influence P450 folding. With predicted structures available for many mammalian P450s involved in metabolism of xenobiotics, it is possible to record allelic variation relative to catalytically important regions in the overall P450 structure and to predict functionally critical differences. Together with information on the relative levels of allelic variant transcripts, comprehensive characterization of the mechanisms that modulate metabolism of natural and synthetic xenobiotics in insects can yield insights into plant-insect coevolution and into novel approaches for chemical pest management.

Bioelectronic Delivery of Electrons to Cytochrome P450 Enzymes

Cytochrome P450s (cyt P450s) are the major oxidative enzymes in human oxidative metabolism of drugs and xenobiotic chemicals. In nature, the iron heme cyt P450s utilize oxygen and electrons delivered from NADPH by a reductase enzyme to oxidize substrates stereo- and regioselectively. Significant research has been directed toward achieving these events electrochemically. This Feature Article discusses the direct electrochemistry of cyt P450s in thin films and the utilization of such films for electrochemically driven biocatalysis. Maintaining and confirming structural integrity and catalytic activity of cyt P450s in films is an essential feature of these efforts. We highlight here our efforts to elucidate the influence of iron heme spin state and secondary structure of human cyt P450s on voltammetric and biocatalytic properties, using methodologies to quantitatively describe the dynamics of these processes in thin films. We also describe the first cyt P450/reductase films that accurately mimic the natural biocatalytic pathway and show how they can be used with voltammetry to elucidate key mechanistic features. Such bioelectronic cyt P450 systems have high value for future drug development, toxicity screening, fundamental investigations, and chemical synthesis systems.

ChemInform Abstract: Role of the Heme Active Site and Protein Environment in Structure, Spectra, and Function of the Cytochrome P450s

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Crystal Structure of OxyC, a Cytochrome P450 Implicated in an Oxidative C–C Coupling Reaction during Vancomycin Biosynthesis

Gene inactivation studies point to the involvement of OxyC in catalyzing the last oxidative phenol coupling reaction during glycopeptide antibiotic biosynthesis. Presently, the substrate and exact timing of the OxyC reaction are unknown. The substrate might be the bicyclic heptapeptide or a thioester derivative bound to a protein carrier domain. OxyC from the vancomycin producer Amycolatopsis orientalis was produced in Escherichia coli and crystallized, and its structure was determined to 1.9 A resolution. OxyC gave UV-visible spectra characteristic of a P450-like hemoprotein in the low spin ferric state. After reduction to the ferrous state by dithionite the CO-ligated form gave a 450-nm peak in a UV-difference spectrum. The addition of vancomycin aglycone to OxyC produced type I changes to the UV spectrum. OxyC exhibits the typical P450-fold, with the Cys ligand loop containing the signature sequence FGHGX-HXCLG and Cys-356 being the proximal axial thiolate ligand of the heme iron. The observation of a water molecule bound to the heme iron is consistent with the UV-visible spectra of OxyC indicating a low spin heme. A polyethylene glycol molecule occupying the active site might mimic the bicyclic heptapeptide substrate. Analysis of the structure of Oxy-proteins and other P450s indicates regions that might be involved in binding of the redox partner and possibly the protein carrier domain.

Membrane-Protein Interactions Contribute to Efficient 27-Hydroxylation of Cholesterol by Mitochondrial Cytochrome P450 27A1

Mitochondrial cytochrome P450 27A1 (P450 27A1) catalyzes 27-hydroxylation of cholesterol, the first step in the alternative bile acid biosynthetic pathway. Although several crystal structures of P450s are known, no structural information is available for the mammalian, membrane-bound enzymes involved in the removal of cholesterol from the body. We prepared a three-dimensional model of P450 27A1 based on the structure of P450 BM-3. Conservative and non-conservative mutations were introduced at hydrophobic and positively charged residues in the putative F-G loop and the adjacent helix G (positions 219-237). Subcellular distribution of the mutant P450s expressed in Escherichia coli was used as a measure of membrane-protein interactions. Conservative substitutions of residues located on the surface, according to our model, L219V, L219I, Y220F, F223Y, L224I, R229K, V231L, F234Y, K236R, and R237K, weakened the association of the mutant P450s with the membrane and led to the appearance of up to 21% of P450 27A1 in the bacterial cytosol. It is likely that the mutated side chains are involved in binding to membrane phospholipids. Substitutions in the F-G loop did not significantly affect the K(m) value for cholesterol hydroxylation. However, non-conservative mutants, L219N, Y220A, Y220S, F223A, K226R, and R229A, had significantly impaired catalytic properties, indicating strict requirements for the size and polarity of the side chains at these positions for the catalysis. The results provide insight into the membrane topology of mitochondrial P450s and indicate the importance of membrane-protein interactions in the efficiency of reactions catalyzed by P450 27A1.

Comparative modelling of cytochromes P450

The superfamily of enzymes known as the cytochromes P450 (P450s) comprises a wide-ranging class of proteins with diverse functions. They are known, amongst other things, to be involved in the hormonal regulation of metabolism and reproduction, as well as having a major clinical significance through their association with diseases such as cancer, diabetes and hepatitis. Knowledge of the three-dimensional (3D) structure of a protein gives insight into its function. The 3D structures of P450s are therefore of considerable scientific interest. A number of high-resolution structures of P450s have been determined by X-ray crystallography and studies of these structures have provided valuable insights into the mechanism of these enzymes. Only one of these structures is mammalian and as yet there is no structural information on human P450s in the public domain. Until such a structure is solved it is necessary to employ alternative methods to gain structural insight into how human P450s perform their biological function. Here we report on the use of comparative modelling to predict the structure of human P450s based on knowledge of their amino acid sequences plus the 3D structures of other (not human) P450s. As an illustrative example of these techniques we have modelled the structure of P450 2C5 using five bacterial P450 structures as templates. We examine the importance of selecting suitable templates, obtaining a good amino acid sequence alignment, and evaluating the models generated. To improve the quality of the models an iterative cycle of sequence alignment, model building, and model evaluation is employed. The result is a model with excellent stereochemistry, good amino acid side chain environment properties, and a Cα trace similar to the crystal structure.

Characterization of the oxygenated intermediate of the thermophilic cytochrome P450 CYP119

Using UV–Vis, resonance Raman, and EPR spectroscopy we have studied the properties of the oxygenated ferrous cytochrome P450 from Sulfolobus Solfataricus, (CYP119). The recently determined crystal structure of CYP119 is compared with other available structures of P450s, and detailed structural and spectroscopic analyses are reported. With several structural similarities to CYP102, such as in-plane iron position and a shorter iron-proximal ligand bond, CYP119 shows low-spin conformation preference in the ferric form and partially in the ferrous form at low temperatures. These structural features can explain the fast autoxidation of the oxyferrous complex of CYP119. Finally, we report the first UV–Vis and EPR spectra of the cryoradiolytically reduced oxygenated intermediate of CYP119. The primary reduced intermediate, a hydroperoxo–ferric complex of CYP119, undergoes a ‘peroxide shunt’ pathway during gradual annealing at 170–195 K and returns to the low-spin ferric form.

Role of the heme active site and protein environment in structure, spectra, and function of the cytochrome p450s.

Role of the heme active site and protein environment in structure, spectra, and function of the cytochrome p450s.

Computer modeling of 3D structures of cytochrome P450s

The understanding of structure-function relationship of enzymes requires detailed information of their three-dimensional structure. Protein structure determination by X-ray and NMR methods, the two most frequently used experimental procedures, are often difficult and time-consuming. Thus computer modeling of protein structures has become an increasingly active and attractive option for obtaining predictive models of three-dimensional protein structures. Specifically, for the ubiquitous metabolizing heme proteins, the cytochrome P450s, the X-ray structures of four isozymes of bacterial origin, P450cam, P450terp, P450BM-3 and P450eryF have now been determined. However, attempts to obtain the structure of mammalian forms by experimental means have thus far not been successful. Thus, there have been numerous attempts to construct models of mammalian P450s using homology modeling methods in which the known structures have been used to various extents and in various strategies to build models of P450 isozymes. In this paper, we review these efforts and then describe a strategy for structure building and assessment of 3D models of P450s recently developed in our laboratory that corrects many of the weaknesses in the previous procedures. The results are 3D models that for the first time are stable to unconstrained molecular dynamics stimulations. The use of this method is demonstrated by the construction and validation of a 3D model for rabbit liver microsomal P450 isozyme 2B4, responsible for the oxidative metabolism of diverse xenobiotics including widely used inhalation anesthetics. Using this 2B4 model, the substrate access channel, substrate binding site and plausible surface regions for binding with P450 redox partners were identified.

Predicting the cytochrome P450 mediated metabolism of xenobiotics

The cytochrome P450s play a unique role in the metabolism of xenobiotics. Characteristics which allow a vast number of foreign compounds to be metabolized by a limited number of enzymes include broad substrate specificity and broad regioselectivity. Because of their importance in both the metabolism and toxicity of drugs and environmental contaminants, efforts are being made to use computational methods to predict these biotransformation pathways. This review describes the recent progress towards the prediction of the tertiary structures of the various P450s and the determination of the electronic characteristics of substrates which determine their tendency to be oxidized by the P450s.

CDNA-expressed human cytochrome P450s: a new age of molecular toxicology and human risk assessment

It has long been recognized that large degree of species differences exists among drug and carcinogen metabolizing enzymes. In particular, differences in cytochrome P450s, the principal enzymes of metabolic activation of procarcinogens, are widespread and may determine species and individual susceptibility to cancer causing chemicals. Although species differences in both the regulation and catalytic activities of P450s are quite large, rodent-based systems are mainly used as the means to determine the degree of hazard of environmental pollutants, pesticides, drugs and other environmental chemicals to humans. During recent years, a large effort has been expended on analyzing directly the structure, properties and catalytic activities of P450s from human tissues. In vitro mutagen testing systems, based on activation by human P450s, are being developed that will supplement other test systems in order to more accurately predict human risk to chemical exposure.

Secondary structure and membrane topology of cytochrome P450s

The secondary structure prediction of 19 microsomal cytochrome P450s from two different families was made on the basis of their amino acid sequences. It was shown that there is structural similarity between the heme-binding sites in these enzymes and those in the bacterial P450cam. An average predicted secondary structure of cytochrome P450 proteins with 70% accuracy contains about 46% α-helices, 12% β-sheets, 9% β-turns, and 33% random coils. In the region of residues 35–120 in microsomal P450s two adjacent βαβ-units (the Rossmann domain), were recognized and may be available to interact with the NADPH-cytochrome P450 reductase. Using the procedure for identification of hydrophobic and membrane-associated α-helical segments, only one N-terminal transmembrane anchor was predicted. Also the heme-binding site may include the surface-bound helix. A model for vertebrate microsomal P450s having an amphipathic membrane protein located on the cytoplasmic side of the endoplasmic reticulum membrane, with their active center lying outside or on the bilayer border, is proposed.

Hybrid cytochromes P-450 identify a substrate binding domain in P-450IIC5 and P-450IIC4.

The cytochrome P-450 superfamily of enzymes catalyzes the oxidative metabolism of innumerable lipophilic compounds (e.g., drugs, carcinogens, steroids). Although the three-dimensional structure of a soluble bacterial P-450 (P-450cam) has been solved, little is known about the structures of the membrane-bound mammalian P-450s. Thus, the structural features of these enzymes that determine their multisubstrate specificity are unknown. In this report, we identify a segment of the primary structure of the structurally similar but functionally distinct cytochromes P-450IIC5 and P-450IIC4, which determines the apparent affinity of these cytochromes for the conversion of progesterone into the mineralocorticoid deoxycorticosterone. P-450IIC5 exhibits a greater than 10-fold lower apparent Km than P-450IIC4 for progesterone 21-hydroxylation. Chimeric cDNAs were constructed and expressed in COS-1 cells, which encode hybrids between these enzymes. The hybrid enzymes were assayed for catalytic activity and compared to the parental proteins. A segment of P-450IIC5 was identified that conferred the lower Km of P-450IIC5 to P-450IIC4. Sequential reduction of the length of the exchanged segments led to a hybrid enzyme with a high affinity derived largely from P-450IIC4, which contains three amino acid residues derived from P-450IIC5 clustered between positions 113 and 118. This suggests that this region is part of a substrate binding domain. This region maps by alignment of amino acid sequences to a residue of P-450cam, which has been implicated in substrate binding, suggesting that these segments of the primary structure serve a similar functional role in these two distantly related proteins.

Structure and regulation of P-450s in the rat P450IIA gene subfamily.

The rat P450IIA subfamily was characterized at the protein, cDNA, and gene level. The purified IIA1 and IIA2 P-450s displayed distinct positional specificities toward hydroxylation of the prototype substrate testosterone. The IIA1 and IIA2 genes were also regulated differently during development; IIA1 was expressed within 1 week after birth in both males and females, and was specifically suppressed in males at puberty, while IIA2 was not expressed in females and was activated at puberty in males. The cDNA-deduced amino acid sequences of these enzymes showed 88% similarity with interspersed regions of high and low similarity indicative of former gene conversion events. Both the IIA1 and IIA2 genes contained nine exons.