Adaptations for the oxidation of polycyclic aromatic hydrocarbons exhibited by the structure of human P450 1A2

Microsomal cytochrome P450 family 1 enzymes play prominent roles in xenobiotic detoxication and procarcinogen activation. P450 1A2 is the principal cytochrome P450 family 1 enzyme expressed in human liver and participates extensively in drug oxidations. This enzyme is also of great importance in the bioactivation of mutagens, including the N-hydroxylation of arylamines. P450-catalyzed reactions involve a wide range of substrates, and this versatility is reflected in a structural diversity evident in the active sites of available P450 structures. Here, we present the structure of human P450 1A2 in complex with the inhibitor alpha-naphthoflavone, determined to a resolution of 1.95 A. alpha-Naphthoflavone is bound in the active site above the distal surface of the heme prosthetic group. The structure reveals a compact, closed active site cavity that is highly adapted for the positioning and oxidation of relatively large, planar substrates. This unique topology is clearly distinct from known active site architectures of P450 family 2 and 3 enzymes and demonstrates how P450 family 1 enzymes have evolved to catalyze efficiently polycyclic aromatic hydrocarbon oxidation. This report provides the first structure of a microsomal P450 from family 1 and offers a template to study further structure-function relationships of alternative substrates and other cytochrome P450 family 1 members.     

Versatile capacity of shuffled cytochrome P450s for dye production

DNA family shuffling is a relatively new method of directed evolution used to create novel enzymes in order to improve their existing properties or to develop new features. This method of evolution in vitro has one basic requirement: a high similarity of initial parental sequences. Cytochrome P450 enzymes are relatively well conserved in their amino acid sequences. Members of the same family can have more than 40% of sequence identity at the protein level and are therefore good candidates for DNA family shuffling. These xenobiotic-metabolising enzymes have an ability to metabolise a wide range of chemicals and produce a variety of products including blue pigments such as indigo. By applying the specifically designed DNA family shuffling approach, catalytic properties of cytochrome P450 enzymes were further extended in the chimeric progeny to include a new range of blue colour formations. This mini-review evokes the possibility of exploiting directed evolution of cytochrome P450s and the novel enzymes created by DNA family shuffling for the production of new dyes.     

Exploring CYP1A1 as anticancer target: homology modeling and in silico inhibitor design

Microsomal cytochrome P450 family 1 enzymes has great importance in the bioactivation of mutagens. P450 catalyzed reactions involve a wide range of substrates, and this versatality is reflected in a structural diversity, evident in the active sites of available P450 structures. This structure offers a template to study further structure-function relationships of alternative substrates and other cytochrome P450 family 1 members. In this paper, we document a homology model of CYP P450 1A1 from Homo sapiens, developed on the basis of template crystal structure of human microsomal P450 1a2 in complex with inhibitor (PDB Id: 2HI4). Homology modeling is performed at the programs, both in the commercial and public realms. We tried to explore CYP1A1 as a potential target for anticancer chemotherapy. To gain an insight into the binding of ligands with enzyme, protein-ligand complex was developed by including information about the known ligand as spatial restraints and optimizing the mutual interactions between the ligand and the binding site. Active site characterization and the study for involvement of specific aminoacids in binding with ligand, facilitates structure based inhibitor design. This study should prove useful in the design and development of potential novel anticancer agents. Figure The refined structure of homology model of CYP1A1     

Structural insight into the altered substrate specificity of human cytochrome P450 2A6 mutants

Human P450 2A6 displays a small active site that is well adapted for the oxidation of small planar substrates. Mutagenesis of CYP2A6 resulted in an increased catalytic efficiency for indole biotransformation to pigments and conferred a capacity to oxidize substituted indoles (Wu, Z.-L., Podust, L.M., Guengerich, F.P. J. Biol. Chem. 49 (2005) 41090-41100.). Here, we describe the structural basis that underlies the altered metabolic profile of three mutant enzymes, P450 2A6 N297Q, L240C/N297Q and N297Q/I300V. The Asn297 substitution abolishes a potential hydrogen bonding interaction with substrates in the active site, and replaces a structural water molecule between the helix B'-C region and helix I while maintaining structural hydrogen bonding interactions. The structures of the P450 2A6 N297Q/L240C and N297Q/I300V mutants provide clues as to how the protein can adapt to fit the larger substituted indoles in the active site, and enable a comparison with other P450 family 2 enzymes for which the residue at the equivalent position was seen to function in isozyme specificity, structural integrity and protein flexibility.     

A novel class of self-sufficient cytochrome P450 monooxygenases in prokaryotes

The Bacillus cytochrome P450 BM3 integrates an entire P450 system in one polypeptide and represents a convenient prokaryotic model for microsomal P450s. This self-sufficient class II P450 is also present in actinomycetes and fungi. By genome analysis we have identified additional homologues in the pathogenic species Bacillus anthracis and Bacillus cereus, and in Ralstonia metallidurans. This analysis also revealed a novel class of putative self-sufficient P450s, P450 PFOR, comprising a class I P450 that is related to Rhodococcus erythropolis CYP116, and a phthalate family oxygenase reductase (PFOR) module. P450 PFOR genes are found in a Rhodococcus strain, three pathogenic Burkholderia species and in the R. metallidurans strain that possesses a P450 BM3 homologue. Co-evolution of P450 and reductase domains is apparent in both types of self-sufficient enzymes. The new class of P450 enzymes is of potential interest for various biotechnological applications.     

The co-immobilization of P450-type nitric oxide reductase and glucose dehydrogenase for the continuous reduction of nitric oxide via cofactor recycling

The co-immobilization of enzymes on target surfaces facilitates the development of self-contained, multi-enzyme biocatalytic platforms. This generally entails the co-immobilization of an enzyme with catalytic value in combination with another enzyme that performs a complementary function, such as the recycling of a critical cofactor. In this study, we co-immobilized two enzymes from different biological sources for the continuous reduction of nitric oxide, using epoxide- and carboxyl-functionalized hyper-porous microspheres. Successful co-immobilization of a fungal nitric oxide reductase (a member of the cytochrome P450 enzyme family) and a bacterial glucose dehydrogenase was obtained with the carboxyl-functionalized microspheres, with enzyme activity maintenance of 158% for nitric oxide reductase and 104% for glucose dehydrogenase. The optimal stoichiometric ratio of these two enzymes was subsequently determined to enable the two independent chemical reactions to be catalyzed concomitantly, allowing for near-synchronous cofactor conversion rates. This dual-enzyme system provides a novel research tool with potential for in vitro investigations of nitric oxide, and further demonstrates the successful immobilization of a P450 enzyme with potential application towards the immobilization of other cytochrome P450 enzymes.     

Determinants of thermostability in the cytochrome P450 fold

Cytochromes P450 are found throughout the biosphere in a wide range of environments, serving a multitude of physiological functions. The ubiquity of the P450 fold suggests that it has been co-opted by evolution many times, and likely presents a useful compromise between structural stability and conformational flexibility. The diversity of substrates metabolized and reactions catalyzed by P450s makes them attractive starting materials for use as biocatalysts of commercially useful reactions. However, process conditions impose different requirements on enzymes to those in which they have evolved naturally. Most natural environments are relatively mild, and therefore most P450s have not been selected in Nature for the ability to withstand temperatures above ~ 40 °C, yet industrial processes frequently require extended incubations at much higher temperatures. Thus, there has been considerable interest and effort invested in finding or engineering thermostable P450 systems. Numerous P450s have now been identified in thermophilic organisms and analysis of their structures provides information as to mechanisms by which the P450 fold can be stabilized. In addition, protein engineering, particularly by directed or artificial evolution, has revealed mutations that serve to stabilize particular mesophilic enzymes of interest. Here we review the current understanding of thermostability as it applies to the P450 fold, gleaned from the analysis of P450s characterized from thermophilic organisms and the parallel engineering of mesophilic forms for greater thermostability. We then present a perspective on how this information might be used to design stable P450 enzymes for industrial application. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.     

Cholesterol Ester Oxidation by Mycobacterial Cytochrome P450

Mycobacteria share a common cholesterol degradation pathway initiated by oxidation of the alkyl side chain by enzymes of cytochrome P450 (CYP) families 125 and 142. Structural and sequence comparisons of the two enzyme families revealed two insertions into the N-terminal region of the CYP125 family (residues 58–67 and 100–109 in the CYP125A1 sequence) that could potentially sterically block the oxidation of the longer cholesterol ester molecules. Catalytic assays revealed that only CYP142 enzymes are able to oxidize cholesteryl propionate, and although CYP125 enzymes could oxidize cholesteryl sulfate, they were much less efficient at doing so than the CYP142 enzymes. The crystal structure of CYP142A2 in complex with cholesteryl sulfate revealed a substrate tightly fit into a smaller active site than was previously observed for the complex of CYP125A1 with 4-cholesten-3-one. We propose that the larger CYP125 active site allows for multiple binding modes of cholesteryl sulfate, the majority of which trigger the P450 catalytic cycle, but in an uncoupled mode rather than one that oxidizes the sterol. In contrast, the more unhindered and compact CYP142 structure enables enzymes of this family to readily oxidize cholesteryl esters, thus providing an additional source of carbon for mycobacterial growth.     

A novel intermediate in the reaction of seleno CYP119 with m-chloroperbenzoic acid

Cytochrome P450-mediated monooxygenation generally proceeds via a reactive ferryl intermediate coupled to a ligand radical [Fe(IV)═O]+? termed Compound I (Cpd I). The proximal cysteine thiolate ligand is a critical determinant of the spectral and catalytic properties of P450 enzymes. To explore the effect of an increased level of donation of electrons by the proximal ligand in the P450 catalytic cycle, we recently reported successful incorporation of SeCys into the active site of CYP119, a thermophilic cytochrome P450. Here we report relevant physical properties of SeCYP119 and a detailed analysis of the reaction of SeCYP119 with m-chloroperbenzoic acid. Our results indicate that the selenolate anion reduces rather than stabilizes Cpd I and also protects the heme from oxidative destruction, leading to the generation of a new stable species with an absorbance maximum at 406 nm. This stable intermediate can be returned to the normal ferric state by reducing agents and thiols, in agreement with oxidative modification of the selenolate ligand itself. Thus, in the seleno protein, the oxidative damage shifts from the heme to the proximal ligand, presumably because (a) an increased level of donation of electrons more efficiently quenches reactive species such as Cpd I and (b) the protection of the thiolate ligand provided by the protein active site structure is insufficient to shield the more oxidizable selenolate ligand.     

Herbivore‐induced poplar cytochrome P450 enzymes of the CYP71 family convert aldoximes to nitriles which repel a generalist caterpillar

Numerous plant species emit volatile nitriles upon herbivory, but the biosynthesis as well as the relevance of these nitrogenous compounds in plant–insect interactions remains unknown. Populus trichocarpa has been shown to produce a complex blend of nitrogenous volatiles, including aldoximes and nitriles, after herbivore attack. The aldoximes were previously reported to be derived from amino acids by the action of cytochrome P450 enzymes of the CYP79 family. Here we show that nitriles are derived from aldoximes by another type of P450 enzyme in P. trichocarpa. First, feeding of deuterium‐labeled phenylacetaldoxime to poplar leaves resulted in incorporation of the label into benzyl cyanide, demonstrating that poplar volatile nitriles are derived from aldoximes. Then two P450 enzymes, CYP71B40v3 and CYP71B41v2, were characterized that produce aliphatic and aromatic nitriles from their respective aldoxime precursors. Both possess typical P450 sequence motifs but do not require added NADPH or cytochrome P450 reductase for catalysis. Since both enzymes are expressed after feeding by gypsy moth caterpillars, they are likely to be involved in herbivore‐induced volatile nitrile emission in P. trichocarpa. Olfactometer experiments showed that these volatile nitriles have a strong repellent activity against gypsy moth caterpillars, suggesting they play a role in induced direct defense against poplar herbivores.     

A predicted three-dimensional structure of human cytochrome P450: implications for substrate specificity

A three-dimensional structure for human cytochrome P450IA1 was predicted based on the crystal coordinates of cytochrome P450cam from Pseudomonas putida. As there was only 15% residue identity between the two enzymes, additional information was used to establish an accurate sequence alignment that is a prerequisite for model building. Twelve representative eukaryotic sequences were aligned and a net prediction of secondary structure was matched against the known alpha-helices and beta-sheets of P450cam. The cam secondary structure provided a fixed main-chain framework onto which loops of appropriate length from the human P450IA1 structure were added. The model-built structure of the human cytochrome conformed to the requirements for the segregation of polar and nonpolar residues between the core and the surface. The first 44 residues of human cytochrome P450 could not be built into the model and sequence analysis suggested that residues 1-26 formed a single membrane-spanning segment. Examination of the sequences of cytochrome P450s from distinct gene families suggested specific residues that could account for the differences in substrate specificity. A major substrate for P450IA1, 3-methyl-cholanthrene, was fitted into the proposed active site and this planar aromatic molecule could be accommodated into the available cavity. Residues that are likely to interact with the haem were identified. The sequence similarity between 59 eukaryotic enzymes was represented as a dendrogram that in general clustered according to gene family. Until a crystallographic structure is available, this model-building study identifies potential residues in cytochrome P450s important in the function of these enzymes and these residues are candidates for site-directed mutagenesis.     

Selectivity improvement of an azole inhibitor of CYP707A by replacing the monosubstituted azole with a disubstituted azole

The plant growth-retardant uniconazole (UNI), a triazole inhibitor of gibberellin biosynthetic enzyme (CYP701A), inhibits multiple P450 enzymes including ABA 8′-hydroxylase (CYP707A), a key enzyme in ABA catabolism. Azole P450 inhibitors bind to a P450 active site by both coordinating to the heme-iron atom via sp² nitrogen and interacting with surrounding protein residues through a lipophilic region. We hypothesized that poor selectivity of UNI may result from adopting a distinct conformation and orientation for different active sites. Based on this hypothesis, we designed and synthesized novel UNI analogs with a disubstituted azole ring (DSI). These analogs were expected to have higher selectivity than UNI because the added functional group may interact with the active site to restrict orientation of the molecule in the active site. DSI-505ME and DSI-505MZ, which have an imidazolyl group with a methyl 5-acrylate, strongly inhibited recombinant CYP707A3, with no growth-retardant effect.     

Formation of indigo by recombinant mammalian cytochrome P450

The development of bicistronic systems for coexpression of recombinant human cytochrome P450 enzymes (P450s) with their redox partner, NADPH-cytochrome P450 reductase (NPR), has enabled P450 activity to be reconstituted within bacterial cells. During expression of recombinant P450 2E1 and some other forms, we observed the formation of a blue pigment in bacterial cultures. The pigment was extracted from cultures and shown to comigrate with standard indigo on TLC. UV-visible spectroscopy and mass spectrometric analysis provided further support for identification of the pigment as indigo. Indigo is known to form following the spontaneous oxidation of 3-hydroxyindole. Accordingly, we speculated that indole, formed as a breakdown product of tryptophan in bacteria, was hydroxylated by the P450 system, leading to indigo formation. Bacterial membranes containing recombinant P450 2E1 and human NPR were incubated in vitro with indole and shown to catalyze formation of a blue pigment in a time- and cofactor-dependent manner. These studies suggest potential applications of mammalian P450 enzymes in industrial indigo production or in the development of novel colorimetric assays based on indole hydroxylation.     

A single mutation in cytochrome P450 BM3 induces the conformational rearrangement seen upon substrate binding in the wild-type enzyme

The multidomain fatty-acid hydroxylase flavocytochrome P450 BM3 has been studied as a paradigm model for eukaryotic microsomal P450 enzymes because of its homology to eukaryotic family 4 P450 enzymes and its use of a eukaryotic-like diflavin reductase redox partner. High-resolution crystal structures have led to the proposal that substrate-induced conformational changes lead to removal of water as the sixth ligand to the heme iron. Concomitant changes in the heme iron spin state and heme iron reduction potential help to trigger electron transfer from the reductase and to initiate catalysis. Surprisingly, the crystal structure of the substrate-free A264E heme domain mutant reveals the enzyme to be in the conformation observed for substrate-bound wild-type P450, but with the iron in the low-spin state. This provides strong evidence that the spin-state shift observed upon substrate binding in wild-type P450 BM3 not only is caused indirectly by structural changes in the protein, but is a direct consequence of the presence of the substrate itself, similar to what has been observed for P450cam. The crystal structure of the palmitoleate-bound A264E mutant reveals that substrate binding promotes heme ligation by Glu(264), with little other difference from the palmitoleate-bound wild-type structure observable. Despite having a protein-derived sixth heme ligand in the substrate-bound form, the A264E mutant is catalytically active, providing further indication for structural rearrangement of the active site upon reduction of the heme iron, including displacement of the glutamate ligand to allow binding of dioxygen.     

Characterisation of two self-sufficient CYP102 family monooxygenases from Ktedonobacter racemifer DSM44963 which have new fatty acid alcohol product profiles

BackgroundTwo self-sufficient CYP102 family encoding genes (Krac_0936 and Krac_9955) from the bacterium Ktedonobacter racemifer DSM44963, which possesses one of the largest bacterial genomes, have been identified.MethodsPhylogenetic analysis of both the encoded cytochrome P450 enzymes, Krac0936 and Krac9955. Both enzymes were produced and their turnovers with fatty acid substrates assessed in vitro and using a whole-cell oxidation system.ResultsKrac0936 hydroxylated straight chain, saturated fatty acids predominantly at the ω-1 and ω-2 positions using NADPH as the cofactor. Krac0936 was less active towards shorter unsaturated fatty acids but longer unsaturated acids were efficiently oxidised. cis,cis-9,12-Octadecadienoic and pentadecanoic acids were the most active substrates tested with Krac0936. Unusually Krac9955 showed very low levels of NAD(P)H oxidation activity though coupling of the reducing equivalents to product formation was high. The product distribution of tridecanoic, tetradecanoic and pentadecanoic acid oxidation by Krac9955 favoured oxidation at the ω-4, ω-5 and ω-6 positions, respectively.ConclusionKrac0936 and Krac9955 are self-sufficient P450 monooxygenases. Krac0936 has a preference for pentadecanoic acid over other straight chain fatty acids and showed little or no activity with dodecanoic or octadecanoic acids. Krac9955 preferably oxidised shorter fatty acids compared to Krac0936 with tridecanoic having the highest levels of product formation. Unlike Krac0936 and P450Bm3, Krac9995 showed lower activities with unsaturated fatty acids.General significanceIn this study of two of the CYP enzymes from K. racemifer we have shown that this bacterium from the Chloroflexi phylum contains genes which encode new proteins with novel activity.     

Cytochrome P450: major player in reperfusion injury

While attention has historically focused on mitochondria as the primary source of ROS in myocardial ischemia/reperfusion injury, recent evidence has implicated cytochrome P450 monooxygenases (CYPs) as a significant factor. CYPs represent a large family of enzymes that catalyze the oxidation of endogenous and exogenous compounds. They catalyze arachidonic acid oxidation to a variety of biologically active eicosanoids that regulate ion channels and protein kinases, with effects on vasomotor tone and cardiac inotropy. They also represent a significant source of reactive oxygen species that may target cellular homeostatic mechanisms and mitochondria. In this review, we will consider the contribution of cytochrome P450 enzymes to reperfusion injury and will speculate on whether the mechanism of injury is due to CYP-mediated ROS production or arachidonic acid metabolites.     

Identification and location of alpha-helices in mammalian cytochromes P450

A model of the alpha-helical structure of mammalian cytochromes P450 is proposed. The location and sequence of alpha-helices in mammalian cytochromes P450 were predicted from their homology with those of cytochrome P450cam, and these sequences were generally confirmed as helical in nature by using a secondary structure prediction method. These analyses were applied to 26 sequences in 6 gene families of cytochrome P450. Mammalian cytochromes P450 consist of approximately 100 amino acid residues more than cytochrome P450cam. This difference was accounted for by three major areas of insertion: (1) at the N-terminus, (2) between helices C and D and between helices D and E, and (3) between helices J and K. Insertion 1 has been suggested by others as a membrane anchoring sequence, but the apparent insertions at 2 and 3 are novel observations; it is suggested that they may be involved in the binding of cytochrome P450 reductase. Only the mitochondrial cytochrome P450 family appeared to show a major variation from this pattern, as insertion 2 was absent, replaced by an insertion between helices G and H and between helices H and I. This may reflect the difference in electron donor proteins that bind to members of this cytochrome P450 family. Other than these differences the model of mammalian cytochromes P450 proposed maintains the general structure of cytochrome P450cam as determined by its alpha-helical composition.     

Cytochrome P450 monooxygenases: an update on perspectives for synthetic application

Cytochrome P450 monooxygenases (P450s) are versatile biocatalysts that catalyze the regio- and stereospeci?c oxidation of non-activated hydrocarbons under mild conditions, which is a challenging task for chemical catalysts. Over the past decade impressive advances have been achieved via protein engineering with regard to activity, stability and specificity of P450s. In addition, a large pool of newly annotated P450s has attracted much attention as a source for novel biocatalysts for oxidation. In this review we give a short up-to-date overview of recent results on P450 engineering for technical applications including aspects of whole-cell biocatalysis with engineered recombinant enzymes. Furthermore, we focus on recently identified P450s with novel biotechnologically relevant properties.