Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent monooxygenases.

The fah1 mutant of Arabidopsis is defective in the accumulation of sinapic acid-derived metabolites, including the guaiacyl-syringyl lignin typical of angiosperms. Earlier results indicated that the FAH1 locus encodes ferulate-5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase (P450) of the general phenylpropanoid pathway. We have cloned the gene encoding this P450 by T-DNA tagging and have confirmed the identity of the cloned gene by complementation of the mutant phenotype. F5H shows 34% amino acid sequence identity with the avocado ripening-induced P450 CYP71A1 and 32% identity with the flavonoid-3',5'-hydroxylases of Petunia hybrida. In contrast, it shares much less homology with cinnamate-4-hydroxylase, a P450 that catalyzes the hydroxylation of cinnamic acid three steps earlier in the general phenylpropanoid pathway. Since the highest degree of identity between F5H and previously sequenced P450s is only 34%, F5H identifies a new P450 subfamily that has been designated CYP84.

Different functions for the interleukin 8 receptors (IL-8R) of human neutrophil leukocytes: NADPH oxidase and phospholipase D are activated through IL-8R1 but not IL-8R2.

Two monoclonal antibodies, anti-IL8R1 and anti-IL8R2, raised against both interleukin 8 receptors (IL-8R) of human neutrophils, IL-8R1 and IL-8R2, were used to study individual receptor functions after stimulation with IL-8, GRO alpha, or NAP-2. Efficacy and selectivity of the antibodies were tested in Jurkat cells transfected with cDNA coding for one or the other receptor. The binding of 125 I labeled IL-8 and IL-8-induced changes of the cytosolic free Ca2+ concentration were inhibited by anti-IL8RI in cells expressing IL-8R1 and by anti-IL8R2 in cells expressing IL-8R2. In human neutrophils, release of elastase was observed after stimulation with IL-8 or GRO alpha. The response to IL-8 was inhibited slightly by anti-IL8R1 and more substantially when both monoclonal antibodies were present, while the response to GRO alpha was inhibited by anti-IL8R2 but was not affected by anti-IL8R1. These results indicate that both IL-8 receptors can signal independently for granule enzyme release. Superoxide production, a measure of the respiratory burst, was obtained with increasing concentrations of IL-8 with maximum effects at 25 to 50 nM, but no response was observed upon challenge with GRO alpha or NAP-2 up to 1000 nM. The superoxide production induced by IL-8 was inhibited by anti-IL8R1, but was not affected by anti-IL8R2. Stimulation of neutrophils with IL-8, in contrast to GRO alpha or NAP-2, also elicited phospholipase D activity. The effect of IL-8 was again inhibited by anti-IL-8R1 but not by anti-IL8R2, indicating that this response, like the respiratory burst, was mediated by IL-8R1. Taken together, our results show that IL-8R1 and IL-8R2 are functionally different. Responses, such as cytosolic free Ca2+ changes and the release of granule enzymes, are mediated through both receptors, whereas the respiratory burst and the activation of phospholipase D depend exclusively on stimulation through IL-8R1.

Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4.

Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.

A mutant cytochrome b5 with a lengthened membrane anchor escapes from the endoplasmic reticulum and reaches the plasma membrane.

Many resident membrane proteins of the endoplasmic reticulum (ER) do not have known retrieval sequences. Among these are the so-called tail-anchored proteins, which are bound to membranes by a hydrophobic tail close to the C terminus and have most of their sequence as a cytosolically exposed N-terminal domain. Because ER tail-anchored proteins generally have short (< or = 17 residues) hydrophobic domains, we tested whether this feature is important for localization, using cytochrome b5 as a model. The hydrophobic domain of cytochrome b5 was lengthened by insertion of five amino acids (ILAAV), and the localization of the mutant was analyzed by immunofluorescence in transiently transfected mammalian cells. While the wild-type cytochrome was localized to the ER, the mutant was relocated to the surface. This relocation was not due to the specific sequence introduced, as demonstrated by the ER localization of a second mutant, in which the original length of the membrane anchor was restored, while maintaining the inserted ILAAV sequence. Experiments with brefeldin A and with cycloheximide demonstrated that the extended anchor mutant reached the plasma membrane by transport along the secretory pathway. We conclude that the short membrane anchor of cytochrome b5 is important for its ER residency, and we discuss the relevance of this finding for other ER tail-anchored proteins.

Synergy between chronic corticosterone and sodium azide treatments in producing a spatial learning deficit and inhibiting cytochrome oxidase activity.

Previously, we developed a rat model of persistent mitochondrial dysfunction based upon the chronic partial inhibition of the mitochondrial enzyme cytochrome oxidase (EC 1.9.3.1). Continuous systemic infusion of sodium azide at approximately 1 mg/kg per hr inhibited cytochrome oxidase activity and produced a spatial learning deficit. In other laboratories, glucocorticoids have been reported to exacerbate neuronal damage from various acute metabolic insults. Therefore, we tested the hypothesis that corticosterone, the primary glucocorticoid in the rat, would potentiate the sodium azide-induced learning deficit. To this end, we first identified nonimpairing doses of sodium azide (approximately 0.75 mg/kg per hr) and corticosterone (100-mg pellet, 3-week sustained-release). We now report that chronic co-administration of these individually nonimpairing treatments produced a severe learning deficit. Moreover, the low dose of corticosterone, which did not elevate serum corticosterone, acted synergistically with sodium azide to inhibit cytochrome oxidase activity. The latter result represents a previously unidentified effect of glucocorticoids that provides a candidate mechanism for glucocorticoid potentiation of neurotoxicity induced by metabolic insult. These results may have the clinical implication of expanding the definition of hypercortisolism in patient populations with compromised oxidative metabolism. Furthermore, they suggest that glucocorticoid treatment may contribute to pathology in disease or trauma conditions that involve metabolic insult.

Stopped-flow NMR spectroscopy: real-time unfolding studies of 6-19F-tryptophan-labeled Escherichia coli dihydrofolate reductase.

Escherichia coli dihydrofolate reductase (DHFR; EC 1.5.1.3) contains five tryptophan residues that have been replaced with 6-19F-tryptophan. The 19F NMR assignments are known in the native, unliganded form and the unfolded form. We have used these assignments with stopped-flow 19F NMR spectroscopy to investigate the behavior of specific regions of the protein in real time during urea-induced unfolding. The NMR data show that within 1.5 sec most of the intensities of the native 19F resonances of the protein are lost but only a fraction (approximately 20%) of the intensities of the unfolded resonances appears. We postulate that the early disappearance of the native resonances indicates that most of the protein rapidly forms an intermediate in which the side chains have considerable mobility. Stopped-flow far-UV circular dichroism measurements indicate that this intermediate retains native-like secondary structure. Eighty percent of the intensities of the NMR resonances assigned to the individual tryptophans in the unfolded state appear with similar rate constants (k approximately 0.14 sec-1), consistent with the major phase of unfolding observed by stopped-flow circular dichroism (representing 80% of total amplitude). These data imply that after formation of the intermediate, which appears to represent an expanded structural form, all regions of the protein unfold at the same rate. Stopped-flow measurements of the fluorescence and circular dichroism changes associated with the urea-induced unfolding show a fast phase (half-time of about 1 sec) representing 20% of the total amplitude in addition to the slow phase mentioned above. The NMR data show that approximately 20% of the total intensity for each of the unfolded tryptophan resonances is present at 1.5 sec, indicating that these two phases may represent the complete unfolding of the two different populations of the native protein.

The three-dimensional structure of NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction.

Quinone reductase [NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2], also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. These two-electron reductions participate in the reductive bioactivation of cancer chemotherapeutic agents such as mitomycin C in tumor cells. Thus, surprisingly, the same enzymatic reaction that protects normal cells activates cytotoxic drugs used in cancer chemotherapy. The 2.1-A crystal structure of rat liver quinone reductase reveals that the folding of a portion of each monomer is similar to that of flavodoxin, a bacterial FMN-containing protein. Two additional portions of the polypeptide chains are involved in dimerization and in formation of the two identical catalytic sites to which both monomers contribute. The crystallographic structures of two FAD-containing enzyme complexes (one containing NADP+, the other containing duroquinone) suggest that direct hydride transfers from NAD(P)H to FAD and from FADH2 to the quinone [which occupies the site vacated by NAD(P)H] provide a simple rationale for the obligatory two-electron reductions involving a ping-pong mechanism.

Cell-free activation of neutrophil NADPH oxidase by a phosphatidic acid-regulated protein kinase.

The phosphorylation-dependent mechanisms regulating activation of the human neutrophil respiratory-burst enzyme, NADPH oxidase, have not been elucidated. We have shown that phosphatidic acid (PA) and diacylglycerol (DG), products of phospholipase activation, synergize to activate NADPH oxidase in a cell-free system. We now report that activation by PA plus DG involves protein kinase activity, unlike other cell-free system activators. NADPH oxidase activation by PA plus DG is reduced approximately 70% by several protein kinase inhibitors [1-(5-isoquinolinesulfonyl)piperazine, staurosporine, GF-109203X]. Similarly, depletion of ATP by dialysis reduces PA plus DG-mediated NADPH oxidase activation by approximately 70%. Addition of ATP, but not a nonhydrolyzable ATP analog, to the dialyzed system restores activation levels to normal. In contrast, these treatments have little effect on NADPH oxidase activation by arachidonic acid or SDS plus DG. PA plus DG induces the phosphorylation of a number of endogenous proteins. Phosphorylation is largely mediated by PA, not DG. A predominant substrate is p47-phox, a phosphoprotein component of NADPH oxidase. Phosphorylation of p47-phox precedes activation of NADPH oxidase and is markedly reduced by the protein kinase inhibitors. In contrast, arachidonic acid alone or SDS plus DG is a poor activator of protein phosphorylation in the cell-free system. Thus, PA induces activation of one or more protein kinases that regulate NADPH oxidase activation in a cell-free system. This cell-free system will be useful for identifying a functionally important PA-activated protein kinase(s) and for dissecting the phosphorylation-dependent mechanisms responsible for NADPH oxidase activation.

Products of Cytochrome P450BioI (CYP107H1)-Catalyzed Oxidation of Fatty Acids

[GRAPHICS] Oxidation of tetradecanoic and hexadecanoic acids by cytochrome P450(Biol) (CYP107H1) produces mainly the 11-, 12-, and 13-hydroxy C-14 fatty acids and the 11- to 15-hydroxy C-16 fatty acids, respectively. In contrast to previous reports, terminal hydroxylation is not observed. The enantiospecificity of fatty acid hydroxylation by P450(Biol) was also determined, and the enzyme was shown to be moderately selective for production of the (R)-alcohols.

The critical role of tryptophan-116 in the catalytic cycle of dimethylsulfoxide reductase from Rhodobacter capsulatus

In dimethylsulfoxide reductase of Rhodobacter capsulatus tryptophan-116 forms a hydrogen bond with a single oxo ligand bound to the molybdenum ion. Mutation of this residue to phenylalanine affected the UV/visible spectrum of the purified Mo-VI form of dimethylsulfoxide reductase resulting in the loss of the characteristic transition at 720 nm. Results of steady-state kinetic analysis and electrochemical studies suggest that tryptophan 116 plays a critical role in stabilizing the hexacoordinate monooxo Mo-VI form of the enzyme and prevents the formation of a dioxo pentacoordinate Mo-VI species, generated as a consequence of the dissociation of one of the dithiolene ligands of the molybdopterin cofactor from the Mo ion. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Acute cadmium chloride administration induces hepatic and renal cytochrome P450 2A5 in the mouse

Modulation of the cytochrome P450 (CYP) monooxygenase system by cadmium was investigated in male, adult DBA/2J mice treated with a single dose (16 Amol/kg body weight, i.p.) of cadmium chloride (CdCl2) at various time points. The total CYP content of kidney microsomes started to decrease 4 hours earlier than in the liver (P < 0.05), with maximal decreases at 24 hours of 56% and 85% in the liver and kidney, respectively. In contrast, both hepatic and renal coumarin 7-hydroxylase (COH) activity (indicative of CYP2A5 activity) relative to total CYP content started to progressively increase at 8 hours, with renal activity 61 times higher than the hepatic activity. Maximum increases were observed, 15-fold in the liver and 64-fold in the kidney after 24 hours. Liver and kidney CYP2A5 mRNA levels increased maximally 12 and 4 hours after treatment, respectively and decreased to almost half 6 hours later. In contrast, kidney and liver CYP2A5 protein levels increased maximally at 18 and 24 hours. This study demonstrates that hepatic and renal CYP2A5 is upregulated by cadmium with a faster response in the kidney than in the liver. This observation is concordant with the fact that kidney is the target organ for cadmium toxicity. The observed increase in the mRNA but not in protein levels after maximal induction suggests involvement of post-transcriptional mechanisms in the regulation of CYP2A5 expression by cadmium.

Modified nicotine metabolism in transgenic tobacco plants expressing the human cytochrome P450 2A6 cDNA

In this study, the human cytochrome P450 (CYP) 2A6 was used in order to modify the alkaloid production of tobacco plants. The cDNA for human CYP2A6 was placed under the control of the constitutive 35S promoter and transferred into Nicotiana tabacum via Agrobacterium-mediated transformation. Transgenic plants showed formation of the recombinant CYP2A6 enzyme but no obvious phenotypic changes. Unlike wild-type tobacco, the transgenic plants accumulated cotinine, a metabolite which is usually formed from nicotine in humans. This result substantiates that metabolic engineering of the plant secondary metabolism via mammalian P450 enzymes is possible in vivo. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Direct electrochemistry of enzymes from the cytochrome P4502C family

The human cytochromes P450 are responsible for the clearance of similar to 90% of xenobiotics yet comparatively little is known about their electrochemistry. Here we report the first direct electrochemistry of P450s from the 2C subfamily; one of the major groups of enzymes from this family. Specifically, the proteins that we have examined are recombinant human P450s 2C9, 2C 18 and 2C 19 and reversible Fe-III/II couples are seen in the absence of dioxygen. Even in the presence of trace amounts of dioxygen, a pronounced cathodic response is seen which is assigned to catalytic reduction of the bound dioxygen ligand by the ferrous P450. (c) 2005 Elsevier B.V. All rights reserved.