Nitric-oxide synthase: a cytochrome P450 family foster child

Nitric-oxide synthase (NOS), the enzyme responsible for mammalian NO generation, is no cytochrome P450, but there are striking similarities between both enzymes. First and foremost, both are heme-thiolate proteins, employing the same prosthetic group to perform similar chemistry. Moreover, they share the same redox partner, a diflavoprotein reductase, which in the case of NOS is incorporated with the oxygenase in one polypeptide chain. There are, however, also conspicuous differences, such as the presence in NOS of the additional cofactor tetrahydrobiopterin, which is applied as an auxiliary electron donor to prevent decay of the oxyferrous complex to ferric heme and superoxide. In this review similarities and differences between NOS and cytochrome P450 are analyzed in an attempt to explain why NOS requires BH4 and why NO synthesis is not catalyzed by a member of the cytochrome P450 family.     

Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase

The molecular characterization of CYP72A1 from Catharanthus roseus (Madagascar periwinkle) was described nearly a decade ago, but the enzyme function remained unknown. We now show by in situ hybridization and immunohistochemistry that the expression in immature leaves is epidermis-specific. It thus follows the pattern previously established for early enzymes in the pathway to indole alkaloids, suggesting that CYP72A1 may be involved in their biosynthesis. The early reactions in that pathway, i.e. from geraniol to strictosidine, contain several candidates for P450 activities. We investigated in this work two reactions, the conversion of 7-deoxyloganin to loganin (deoxyloganin 7-hydroxylase, DL7H) and the oxidative ring cleavage converting loganin into secologanin (secologanin synthase, SLS). The action of DL7H has not been demonstrated in vitro previously, and SLS has only recently been identified as P450 activity in one other plant. We show for the first time that both enzyme activities are present in microsomes from C. roseus cell cultures. We then tested whether CYP72A1 expressed in E. coli as a translational fusion with the C. roseus P450 reductase (P450Red) has one or both of these activities. The results show that CYP72A1 converts loganin into secologanin.     

Nitric oxide synthase is a cytochrome P-450 type hemoprotein.

Nitric oxide has emerged as an important mammalian metabolic intermediate involved in critical physiological functions such as vasodilation, neuronal transmission, and cytostasis. Nitric oxide synthase (NOS) catalyzes the five-electron oxidation of L-arginine to citrulline and nitric oxide. Cosubstrates for the reaction include molecular oxygen and NADPH. In addition, there is a requirement for tetrahydrobiopterin. NOS also contains the coenzymes FAD and FMN and demonstrates significant amino acid sequence homology to NADPH-cytochrome P-450 reductase. Herein we report the identification of the inducible macrophage NOS as a cytochrome P-450 type hemoprotein. The pyridine hemochrome assay showed that the NOS contained a bound protoporphyrin IX heme. The reduced carbon monoxide binding spectrum shows an absorption maximum at 447 nm indicative of a cytochrome P-450 hemoprotein. A mixture of carbon monoxide and oxygen (80%/20%) potently inhibited the reaction (73-79%), showing that the heme functions directly in the oxidative conversion of L-arginine to nitric oxide and citrulline. Additionally, partially purified NOS from rat cerebellum was inhibited by CO, suggesting that this isoform may also contain a P-450-type heme. NOS is the first example of a soluble cytochrome P-450 in eukaryotes. In addition, the presence of FAD and FMN indicates that this is the first catalytically self-sufficient mammalian P-450 enzyme, containing both a reductase and a heme domain on the same polypeptide.     

Screening method for cDNAs encoding putative enzymes converting loganin into secologanin by a transgenic yeast culture

A screening method was developed for the detection of enzymes converting loganin to secologanin, a precursor in the biosynthesis of indole alkaloids. The method uses a transgenic yeast culture expressing two cDNAs encoding enzymes involved in the terpenoid indole alkaloid biosynthesis. In the presence of secologanin, the yeast culture produces a yellow compound visible on nitrocellulose. This color change was used to screen a cDNA library of Catharanthus roseus for a putative enzyme converting loganin into secologanin.     

Molecular cloning and biochemical characterization of a novel cytochrome P450, flavone synthase II, that catalyzes direct conversion of flavanones to flavones

Cytochrome P450 cDNAs, AFNS2 and TFNS5, were isolated from snapdragon and torenia petal cDNA libraries, respectively, based on the sequence homology with licorice CYP93B1 cDNA encoding (2S)-flavanone 2-hydroxylase. They were expressed in yeast and identified to encode flavone synthase II catalyzing direct conversion of flavanones to flavones probably via 2-hydroxyflavanones.     

Radioimmunoassay for the determination of loganin and the biotransformation of loganin to secologanin by plant cell cultures

A radioimmunoassay technique has been developed for the quantitative measurement of loganin in crude extracts from both fresh and dried material of whole plants and cultivated plant cells. The assay makes use of 6′-carboxyloganin which is rendered immunogenic through linkage to bovine serum albumin. The tracer molecule was synthesized via periodate opening of the glucose moiety of loganin and subsequent reduction with sodium borotritide of high specific activity. The rabbit antibodies had a high affinity (K_a = 1.6 × 10⁹1/mol) for loganin and permitted the detection of as little as 0.1 ng per 0.05 ml of sample. The antiserum was highly specific for loganin and its aglycone, with only 10-hydroxyloganin and 7-epi-loganin showing a substantial cross reactivity. A number of cell cultures of the Caprifoliaceae were tested for their ability to transform added loganin to secologanin. By labelled precursor feeding experiments members of the genera Weigelia, Lonicera, Hydrangea and Symphoricarpus were found to open the cyclopentane ring of loganin. The time course of the biotransformation of loganin was monitored using radioimmunoassays for both loganin and secologanin and cell cultures of Lonicera tatarica as biological material.     

Molecular identity and gene expression of aldosterone synthase cytochrome P450

11Beta-hydroxylase (CYP11B1) of bovine adrenal cortex produced corticosterone as well as aldosterone from 11-deoxycorticosterone in the presence of the mitochondrial P450 electron transport system. CYP11B1s of pig, sheep, and bullfrog, when expressed in COS-7 cells, also performed corticosterone and aldosterone production. Since these CYP11B1s are present in the zonae fasciculata and reticularis as well as in the zona glomerulosa, the zonal differentiation of steroid production may occur by the action of still-unidentified factor(s) on the enzyme-catalyzed successive oxygenations at C11- and C18-positions of steroid. In contrast, two cDNAs, one encoding 11beta-hydroxylase and the other encoding aldosterone synthase (CYP11B2), were isolated from rat, mouse, hamster, guinea pig, and human adrenals. The expression of CYP11B1 gene was regulated by cyclic AMP (cAMP)-dependent signaling, whereas that of CYP11B2 gene by calcium ion-signaling as well as cAMP-signaling. Salt-inducible protein kinase, a cAMP-induced novel protein kinase, was one of the regulators of CYP11B2 gene expression.     

Recognition and oxidative metabolism of cyclodipeptides by hepatic cytochrome P450

Possible recognition of peptide derivatives by hepatic cytochrome P450 3A has been suggested by binding and metabolism of numerous pseudopeptidic compounds such as ergot derivatives and cyclosporin.Natural linear or cyclic dipeptides containing hydrophobic amino acids produced by microorganisms and present in mammals are able to interact with the P450 active site through either iron-amine interactions (Type II) or hydrophobic Type I interactions. P450 3A from dexamethasone-treated rats or yeast-expressed P450 human 3A4 are the most potent in such interactions, which are particularly strong with peptides containing a histidyl residue.Some cyclodipeptides are rapidly transformed by rat cytochrome P450 3A to mono- or dihydroxylated metabolites, with turnovers around 3 nmoles min(-1) P450(-1). Linear peptides are poorly transformed in these conditions. This metabolism of cyclodipeptides occurs in 8 species including man.Such interactions and metabolism have only minor consequences in terms of P450 3A binding and metabolism of classical P450 3A substrates. These data reinforce the concept that, in addition to their effect on the regulation of P450 neosynthesis, naturally occurring endogenous peptides are also substrates of P450 3A. The physiological activities of these peptides may be modulated by their metabolism.     

3-Deoxy-D-manno-octulosonate-8-phosphate synthase catalyzes the C-O bond cleavage of phosphoenolpyruvate

The mechanism of 3-deoxy-D-manno-octulosonate-8-phosphate (KDO8P) synthase was investigated. When [18O]-PEP specifically labeled in the enolic oxygen is a substrate for KDO8P synthase, the 18O is recovered in Pi. This indicates that the KDO8P synthase reaction proceeds with C-O bond cleavage of PEP similar to that observed in the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase catalyzed condensation of PEP and erythrose-4-phosphate (1). No evidence for a covalent enzyme-PEP intermediate could be obtained. No [32P]-Pi exchange into PEP nor scrambling of bridge 18O to non-bridging positions in [18O]-PEP was observed in the presence or absence of arabinose-5-phosphate or its analog ribose-5-phosphate. Bromopyruvate inactivated KDO8P synthase in a time dependent process. It is likely that bromopyruvate reacts with a functional group at the PEP binding site since PEP, but not arabinose-5-phosphate, protects against inactivation.     

Structural characterization of the gene and corresponding cDNA for the cytochrome P450rm from Rhodotorula minuta which catalyzes formation of isobutene and 4-hydroxylation of benzoate

Cytochrome P450rm was previously isolated from the basidiomycete yeast Rhodotorula minuta as a bifunctional enzyme with isobutene-forming and benzoate 4-hydroxylase activities. We cloned the gene and corresponding cDNA for P450rm in order to characterize the enzyme in the context of fungal phylogeny and physiology. From the cDNA sequence, P450rm was deduced to have 527 amino acids with a calculated molecular weight of 59 136. P450rm shared 48% amino acid sequence identity with CYP53A1 from Aspergillus niger, indicating that the gene belongs to a novel subfamily of CYP53, CYP53B. However, the organization of the P450rm gene, which has eight exons and seven introns, differed completely to that of CYP53A1. Northern analysis demonstrated that the level of P450rm mRNA expression increased when L-phenylalanine was used as sole carbon source. These results suggest that P450rm has been well conserved during the evolution of fungi as a benzoate 4-hydroxylase in the dissimilation pathway starting from L-phenylalanine     

Why human cytochrome P450c21 is a progesterone 21-hydroxylase

Human cytochrome P450c21 (steroid 21-hydroxylase, CYP21A2) catalyzes the 21-hydroxylation of progesterone (P4) and its preferred substrate 17α-hydroxyprogestrone (17OHP4). CYP21A2 activities, which are required for cortisol and aldosterone biosynthesis, involve the formation of energetically disfavored primary carbon radicals. Therefore, we hypothesized that the binding of P4 and 17OHP4 to CYP21A2 restricts access of the reactive heme-oxygen complex to the C-21 hydrogen atoms, suppressing oxygenation at kinetically more favorable sites such as C-17 and C-16, which are both hydroxylated by cytochrome P450c17 (CYP17A1). We reasoned that expansion of the CYP21A2 substrate-binding pocket would increase substrate mobility and might yield additional hydroxylation activities. We built a computer model of CYP21A2 based principally on the crystal structure of CYP2C5, which also 21-hydroxylates P4. Molecular dynamics simulations indicate that binding of the steroid nucleus perpendicular to the plane of the CYP21A2 heme ring limits access of the heme oxygen to the C-21 hydrogen atoms. Residues L107, L109, V470, I471, and V359 were found to contribute to the CYP21A2 substate-binding pocket. Mutation of V470 and I471 to alanine or glycine preserved P4 21-hydroxylase activity, and mutations of L107 or L109 were inactive. Mutations V359A and V359G, in contrast, acquired 16α-hydroxylase activity, accounting for 40% and 90% of the P4 metabolites, respectively. We conclude that P4 binds to CYP21A2 in a fundamentally different orientation than to CYP17A1 and that expansion of the CYP21A2 substrate-binding pocket allows additional substrate trajectories and metabolic switching.     

Arabidopsis cytochrome P450 monooxygenase 71A13 catalyzes the conversion of indole-3-acetaldoxime in camalexin synthesis

Camalexin (3-thiazol-2-yl-indole) is an indole alkaloid phytoalexin produced by Arabidopsis thaliana that is thought to be important for resistance to necrotrophic fungal pathogens, such as Alternaria brassicicola and Botrytis cinerea. It is produced from Trp, which is converted to indole acetaldoxime (IAOx) by the action of cytochrome P450 monooxygenases CYP79B2 and CYP79B3. The remaining biosynthetic steps are unknown except for the last step, which is conversion of dihydrocamalexic acid to camalexin by CYP71B15 (PAD3). This article reports characterization of CYP71A13. Plants carrying cyp71A13 mutations produce greatly reduced amounts of camalexin after infection by Pseudomonas syringae or A. brassicicola and are susceptible to A. brassicicola, as are pad3 and cyp79B2 cyp79B3 mutants. Expression levels of CYP71A13 and PAD3 are coregulated. CYP71A13 expressed in Escherichia coli converted IAOx to indole-3-acetonitrile (IAN). Expression of CYP79B2 and CYP71A13 in Nicotiana benthamiana resulted in conversion of Trp to IAN. Exogenously supplied IAN restored camalexin production in cyp71A13 mutant plants. Together, these results lead to the conclusion that CYP71A13 catalyzes the conversion of IAOx to IAN in camalexin synthesis and provide further support for the role of camalexin in resistance to A. brassicicola.     

Engineering cytochrome c peroxidase into cytochrome P450: a proximal effect on heme-thiolate ligation.

In an effort to investigate factors required to stabilize heme-thiolate ligation, key structural components necessary to convert cytochrome c peroxidase (CcP) into a thiolate-ligated cytochrome P450-like enzyme have been evaluated and the H175C/D235L CcP double mutant has been engineered. The UV-visible absorption, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectra for the double mutant at pH 8.0 are reported herein. The close similarity between the spectra of ferric substrate-bound cytochrome P450cam and those of the exogenous ligand-free ferric state of the double mutant with all three techniques support the conclusion that the latter has a pentacoordinate, high-spin heme with thiolate ligation. Previous efforts to prepare a thiolate-ligated mutant of CcP with the H175C single mutant led to Cys oxidation to cysteic acid [Choudhury et al. (1994) J. Biol. Chem. 267, 25656-25659]. Therefore it is concluded that changing the proximal Asp235 residue to Leu is critical in forming a stable heme-thiolate ligation in the resting state of the enzyme. To further probe the versatility of the CcP double mutant as a ferric P450 model, hexacoordinate low-spin complexes have also been prepared. Addition of the neutral ligand imidazole or of the anionic ligand cyanide results in formation of hexacoordinate adducts that retain thiolate ligation as determined by spectral comparison to the analogous derivatives of ferric P450cam. The stability of these complexes and their similarity to the analogous forms of P450cam illustrates the potential of the H175C/D235L CcP double mutant as a model for ferric P450 enzymes. This study marks the first time a stable cyanoferric complex of a model P450 has been made and demonstrates the importance of the environment around the primary coordination ligands in stabilizing metal-ligand ligation.     

Co-incorporation of heterologously expressed Arabidopsis cytochrome P450 and P450 reductase into soluble nanoscale lipid bilayers

Heterologous expression of CYP73A5, an Arabidopsis cytochrome P450 monooxygenase, in baculovirus-infected insect cells yields correctly configured P450 detectable by reduced CO spectral analysis in microsomes and cell lysates. Co-expression of a housefly NADPH P450 reductase substantially increases the ability of this P450 to hydroxylate trans-cinnamic acid, its natural phenylpropanoid substrate. For development of high-throughput P450 substrate profiling procedures, membrane proteins derived from cells overexpressing CYP73A5 and/or NADPH P450 reductase were incorporated into soluble His(6)-tagged nanoscale lipid bilayers (Nanodiscs) using a simple self-assembly process. Biochemical characterizations of nickel affinity-purified and size-fractionated Nanodiscs indicate that CYP73A5 protein assembled into Nanodiscs in the absence of NADPH P450 reductase maintains its ability to bind its t-cinnamic acid substrate. CYP73A5 protein co-assembled with P450 reductase into Nanodiscs hydroxylates t-cinnamic acid using reduced pyridine nucleotide as an electron source. These data indicate that baculovirus-expressed P450s assembled in Nanodiscs can be used to define the chemical binding profiles and enzymatic activities of these monooxygenases.     

The cytochrome P450 2B4-NADPH cytochrome P450 reductase electron transfer complex is not formed by charge-pairing.

Attempts to covalently link NADPH-cytochrome P450 reductase to cytochrome P450 2B4 using a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylisopropyl)carbodiimide, were unsuccessful, despite the fact that under the same conditions about 30% of P450 2B4 could be covalently linked with cytochrome b5 in a functionally active complex (Tamburini, P. P., and Schenkman, J. B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 11-15). This suggested that the functional electron transfer complex between P450 2B4 and reductase is not stabilized by electrostatic forces. Raising the ionic strength of the medium is disruptive to salt bridges and was used to further test whether P450 2B4 and the reductase form charge-pairing complexes. Instead of inhibiting electron transfer, high ionic strength increased the apparent fast phase rate constant and the fraction of P450 2B4 reduced in the fast phase. The possibility that electron transfer between NADPH-cytochrome P450 reductase and P450 2B4 is diminished by charge repulsion was examined. Consistent with this hypothesis, the Km of P450 2B4 for reductase was decreased 26-fold by increasing the ionic strength from 10 to 100 mM sodium phosphate without affecting the Vmax. The rate of benzphetamine N-demethylation also was increased by elevation of the ionic strength. Electron transfer from the reductase to other charged redox acceptors, e.g. cytochrome c and ferricyanide, was also stimulated by increased ionic strength. However, no similar stimulation was observed with the uncharged acceptor 1,4-benzoquinone. Polylysine, a polypeptide that binds to anionic sites, enhanced electron transfer from NADPH to ferricyanide and the apparent fast phase of reduction of cytochrome P450. The results are consistent with the hypothesis that charges on NADPH-cytochrome P450 reductase and cytochrome P450 decrease the stability of the electron transfer complex.     

Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein reductase (EC, cytochrome P450 reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivo metabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.     

Resveratrol is a selective human cytochrome P450 1A1 inhibitor.

Resveratrol (trans-3,4',5-trihydroxystilbene) is a phytoalexin compound found in juice and wine produced from dark-skinned grape cultivars and reported to have anti-inflammatory and anticarcinogenic activities. To investigate the mechanism of anticarcinogenic activities of resveratrol, the effects on cytochrome P450 (P450) were determined in human liver microsomes and Escherichia coli membranes coexpressing human P450 1A1 or P450 1A2 with human NADPH-P450 reductase (bicistronic expression system). Resveratrol slightly inhibited ethoxyresorufin O-deethylation (EROD) activity in human liver microsomes with an IC(50) of 1.1 mM. Interestingly, resveratrol exhibited potent inhibition of human P450 1A1 in a dose-dependent manner with IC(50) of 23 microM for EROD and IC(50) of 11 microM for methoxyresorufin O-demethylation (MROD). However, the inhibition of human P450 1A2 by resveratrol was not so strong (IC(50) 1.2 mM for EROD and 580 microM for MROD). Resveratrol showed over 50-fold selectivity for P450 1A1 over P450 1A2. The activities of human NADPH-P450 reductase were not significantly changed by resveratrol. In a human P450 1A1/reductase bicistronic expression system, resveratrol inhibited human P450 1A1 activity in a mixed-type inhibition (competitive-noncompetitive) with a K(i) values of 9 and 89 microM. These results suggest that resveratrol is a selective human P450 1A1 inhibitor, and may be considered for use as a strong cancer chemopreventive agent in humans.