Nitrobenzoates and Aminobenzoates Are Chemoattractants for Pseudomonas Strains

Three Pseudomonas strains were tested for the ability to sense and respond to nitrobenzoate and aminobenzoate isomers in chemotaxis assays. Pseudomonas putida PRS2000, a strain that grows on benzoate and 4-hydroxybenzoate by using the β-ketoadipate pathway, has a well-characterized β-ketoadipate-inducible chemotactic response to aromatic acids. PRS2000 was chemotactic to 3- and 4-nitrobenzoate and all three isomers of aminobenzoate when grown under conditions that induce the benzoate chemotactic response. P. putida TW3 and Pseudomonas sp. strain 4NT grow on 4-nitrotoluene and 4-nitrobenzoate by using the ortho (β-ketoadipate) and meta pathways, respectively, to complete the degradation of protocatechuate derived from 4-nitrotoluene and 4-nitrobenzoate. However, based on results of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase assays, both strains were found to use the β-ketoadipate pathway for the degradation of benzoate. Both strains were chemotactic to benzoate, 3- and 4-nitrobenzoate, and all three aminobenzoate isomers after growth with benzoate but not succinate. Strain TW3 was chemotactic to the same set of aromatic compounds after growth with 4-nitrotoluene or 4-nitrobenzoate. In contrast, strain 4NT did not respond to any aromatic acids when grown with 4-nitrotoluene or 4-nitrobenzoate, apparently because these substrates are not metabolized to the inducer (β-ketoadipate) of the chemotaxis system. The results suggest that strains TW3 and 4NT have a β-ketoadipate-inducible chemotaxis system that responds to a wide range of aromatic acids and is quite similar to that present in PRS2000. The broad specificity of this chemotaxis system works as an advantage in strains TW3 and 4NT because it functions to detect diverse carbon sources, including 4-nitrobenzoate.     

Key Aromatic-Ring-Cleaving Enzyme, Protocatechuate 3,4-Dioxygenase, in the Ecologically Important Marine Roseobacter Lineage

Aromatic compound degradation in six bacteria representing an ecologically important marine taxon of the α-proteobacteria was investigated. Initial screens suggested that isolates in the Roseobacter lineage can degrade aromatic compounds via the β-ketoadipate pathway, a catabolic route that has been well characterized in soil microbes. Six Roseobacter isolates were screened for the presence of protocatechuate 3,4-dioxygenase, a key enzyme in the β-ketoadipate pathway. All six isolates were capable of growth on at least three of the eight aromatic monomers presented (anthranilate, benzoate, p-hydroxybenzoate, salicylate, vanillate, ferulate, protocatechuate, and coumarate). Four of the Roseobacter group isolates had inducible protocatechuate 3,4-dioxygenase activity in cell extracts when grown on p-hydroxybenzoate. The pcaGH genes encoding this ring cleavage enzyme were cloned and sequenced from two isolates, Sagittula stellata E-37 and isolate Y3F, and in both cases the genes could be expressed in Escherichia coli to yield dioxygenase activity. Additional genes involved in the protocatechuate branch of the β-ketoadipate pathway (pcaC, pcaQ, and pobA) were found to cluster with pcaGH in these two isolates. Pairwise sequence analysis of the pca genes revealed greater similarity between the two Roseobacter group isolates than between genes from either Roseobacter strain and soil bacteria. A degenerate PCR primer set targeting a conserved region within PcaH successfully amplified a fragment of pcaH from two additional Roseobacter group isolates, and Southern hybridization indicated the presence of pcaH in the remaining two isolates. This evidence of protocatechuate 3,4-dioxygenase and the β-ketoadipate pathway was found in all six Roseobacter isolates, suggesting widespread abilities to degrade aromatic compounds in this marine lineage.     

Molecular Analysis and Heterologous Expression of an Inducible Cytochrome P-450 Protein from Periwinkle (Catharanthus roseus L.)

We screened cDNA libraries from periwinkle (Catharanthus roseus) cell cultures induced for indole alkaloid synthesis and selected clones for induced cytochrome P-450 (P-450) proteins by differential hybridization, size of the hybridizing mRNA, and presence of amino acid motifs conserved in many P-450 families. Four cDNAs satisfying these criteria were analyzed in detail. They were grouped in two classes (pCros1, pCros2) that represented two closely related genes of a new P-450 family designated CYP72. Antiserum against a cDNA fusion protein overexpressed in Escherichia coli recognized in C. roseus a protein band of 56 kD. Quantification of western blots showed that it represented 1.5 ± 0.5 and 6 ± 1 μg/mg of protein in the membranes from noninduced and induced cells, respectively, and analysis of the total P-450 content suggested that the cDNA-encoded protein was one of the dominant P-450 proteins. The pathway to indole alkaloids contains two known P-450 enzymes, geraniol-10-hydroxylase (GE10H) and nerol-10-hydroxylase (NE10H). The induction kinetics of the cloned P-450 protein and of GE10H activity were similar, but those of NE10H were different. Western blots with membranes from other plants suggested that P-450 CYP72 is specific for C. roseus and other plants with GE10H activity. A tentative assignment of CYP72 as GE10H is discussed. The cDNA was recloned for expression in Saccharomyces cerevisiae, and the presence of the protein was demonstrated by western blots. Assays for GE10H failed to detect enzyme activity, and the same negative result was obtained for NE10H and other P-450 enzymes that are present in C. roseus.     

Purification and properties of cytochrome P-450 from Moraxella sp

A cytochrome P-450 has been purified to homogeneity from a Moraxella species that is able to grow on guaiacol as the sole source of carbon and energy. The pure cytochrome was a monomeric protein of about 52 kDa, with no catalytic activity towards guaiacol. The difference in mM extinction coefficients between 450 and 490 nm in the CO-difference spectrum was 89.5 mM-1.cm-1. The typical shift of the Soret band from 415 to 390 nm that is attributed to the high-spin state of the cytochrome was observed in the presence of guaiacol and other 2-alkoxyphenols with up to 5 carbons in the side chain. It was also obtained with anisole. The maximum difference in mM extinction coefficients between 390 and 420 nm in the P-450 + ligand minus P-450 spectrum was 65 mM-1.cm-1 in all instances. The dissociation constants of the complexes formed between the pure protein and various O-alkoxyphenols were measured, and ranged from 0.1 microM (guaiacol) to 24 microM (2-butoxyphenol). The dissociation constants were 1 microM for anisole, and over 90 microM for phenol. Catechol induced no spectral change in cytochrome P-450 and appeared to be a weak inhibitor of guaiacol binding. The same spectral shift as induced by guaiacol was observed at high P-450 concentration over 1 microM in the absence of any added ligand and disappeared after dilution. The reduction of pure P-450 by dithionite was immediate, but became very slow, and was complete after 10 min or more at 25 degrees C in the presence of guaiacol. This effect was also obtained with the 2 isomers, 3- and 4-methoxyphenols, and with metyrapone, an inhibitor of guaiacol binding that induced the low-spin state. Preliminary experiments using the crude cell lysate or a reconstructed system with purified P-450 and a protein fraction indicated NADH-dependent guaiacol degradation. This was in agreement with the former hypothesis of Moraxella P-450 acting as a monooxygenase in the demethylation of guaiacol. However, cis, cis-muconate rather than catechol was obtained from the substrate, most likely a consequence of the potent catechol 1,2-dioxygenase activity present in the non-purified protein fractions used.     

Clinical Isolate of Pseudomonas aeruginosa that Degrades Salicylate by the ortho Pathway

A clinical isolate of Pseudomonas aeruginosa was found capable of utilizing salicylate by the salicylate hydroxylase and β-ketoadipate pathway.     

Degradation of Morpholine by an Environmental Mycobacterium Strain Involves a Cytochrome P-450

A Mycobacterium strain (RP1) was isolated from a contaminated activated sludge collected in a wastewater treatment unit of a chemical plant. It was capable of utilizing morpholine and other heterocyclic compounds, such as pyrrolidine and piperidine, as the sole source of carbon, nitrogen, and energy. The use of in situ ¹H nuclear magnetic resonance (¹H NMR) spectroscopy allowed the determination of two intermediates in the biodegradative pathway, 2-(2-aminoethoxy)acetate and glycolate. The inhibitory effects of metyrapone on the degradative abilities of strain RP1 indicated the involvement of a cytochrome P-450 in the biodegradation of morpholine. This observation was confirmed by spectrophotometric analysis and ¹H NMR. Reduced cell extracts from morpholine-grown cultures, but not succinate-grown cultures, gave rise to a carbon monoxide difference spectrum with a peak near 450 nm, which indicated the presence of a soluble cytochrome P-450. ¹H NMR allowed the direct analysis of the incubation medium containing metyrapone, a specific inhibitor of cytochrome P-450. The inhibition of morpholine degradation was dependent on the morpholine/metyrapone ratio. The heme-containing monooxygenase was also detected in pyrrolidine- and piperidine-grown cultures. The abilities of different compounds to support strain growth or the induction of a soluble cytochrome P-450 were assayed. The results suggest that this enzyme catalyzes the cleavage of the C—N bond of the morpholine ring.     

Biochemical characterization of a spearmint mutant that resembles peppermint in monoterpene content

A radiation-induced mutant of Scotch spearmint (Mentha × gracilis) was shown to produce an essential oil containing principally C3-oxygenated p-menthane monoterpenes that are typical of peppermint, instead of the C6-oxygenated monoterpene family characteristic of spearmint. In vitro measurement of all of the enzymes responsible for the production of both the C3-oxygenated and C6-oxygenated families of monoterpenes from the common precursor (−)-limonene indicated that a virtually identical complement of enzymes was present in wild type and mutant, with the exception of the microsomal, cytochrome P-450-dependent (−)-limonene hydroxylase; the C6-hydroxylase producing (−)-trans-carveol in the wild type had been replaced by a C3-hydroxylase producing (−)-trans-isopiperitenol in the mutant. Additionally, the mutant, but not the wild type, could carry out the cytochrome P-450-dependent epoxidation of the α,β-unsaturated bond of the ketones formed via C3-hydroxylation. Although present in the wild type, the enzymes of the C3-pathway that convert trans-isopiperitenol to menthol isomers are synthetically inactive because of the absence of the key C3-oxygenated intermediate generated by hydroxylation of limonene. These results, which clarify the origins of the C3- and C6-oxygenation patterns, also allow correction of a number of earlier biogenetic proposals for the formation of monoterpenes in Mentha.     

The demethylation of guaiacol by a new bacterial cytochrome P-450

Spectroscopic studies were carried with a cytochrome P-450 in Moraxella sp., strain GU2, that could grow on guaiacol or 2-ethoxyphenol as the sole source of carbon and energy. The dissociation constant of the guaiacol-cytochrome complex was estimated to 0.15 microM, as determined in vivo or using the cell soluble extract. Cytochrome P-450 could also bind 2-ethoxyphenol, 2-propoxyphenol, and 2-butoxyphenol, and the dissociation constants have been determined in each case. Metyrapone depressed the degradation of guaiacol by whole bacteria, and was bound competitively to guaiacol with a constant of about 0.8 mM. Some catechol was excreted by the bacteria when growing on either guaiacol or 2-ethoxyphenol. Catechol and the other product of guaiacol demethylation, formaldehyde, were further oxidized by the bacteria. All the data available so far are consistent with cytochrome P-450 in Moraxella GU2 as a hydroxylase for the guaiacol side chain, behaving as a nonspecific O-dealkylase with broad specificity for guaiacol and homologous compounds with a longer carbon part in the side chain.     

Cytochrome p-450-dependent hydroxylation of lauric Acid at the subterminal position and oxidation of unsaturated analogs in wheat microsomes

Microsomes from etiolated wheat (Triticum aestivum L. cv Etoile de Choisy) shoots catalyzed the reduced nicotinamide adenine dinucleotide phosphate-dependent hydroxylation of lauric acid predominantly at the subterminal or (ω-1) position (65%). Minor amounts of 10-hydroxy- (31%) and 9-hydroxylaurate (4%) were also formed. The reaction was catalyzed by cytochrome P-450, since enzyme activity was strongly inhibited by tetcyclacis, carbon monoxide, and antibodies against NADPH-cytochrome c (P-450)-reductase. The apparent K_m for lauric acid was estimated to be 8.5 ± 2.0 μm. Seed treatment with the safener naphthalic acid anhydride or treatment of seedlings with phenobarbital increased cytochrome P-450 content and lauric acid hydroxylase (LAH) activity of the microsomes. A combination of both treatments further stimulated LAH activity. A series of radiolabeled unsaturated lauric acid analogs (8-, 9-, 10-, and 11-dodecenoic acids) was used to explore the regioselectivity and catalytic capabilities of induced wheat microsomes. It has been found that wheat microsomes catalyzed the reduced nicotinamide adenine dinucleotide phosphate-dependent epoxidation of sp2 carbons concurrently with hydroxylation at saturated positions. The regioselectivity of oxidation of the unsaturated substrates and that of lauric acid were similar. Preincubation of wheat microsomes with reduced nicotinamide adenine dinucleotide phosphate and 11-dodecenoic acid resulted in a partial loss of LAH activity.     

Intermediates in the recycling of 5-methylthioribose to methionine in fruits

The recycling of 5-methylthioribose (MTR) to methionine in avocado (Persea americana Mill, cv Hass) and tomato (Lycopersicum esculentum Mill, cv unknown) was examined. [¹⁴CH₃]MTR was not metabolized in cell free extract from avocado fruit. Either [¹⁴CH₃]MTR plus ATP or [¹⁴CH₃]5-methylthioribose-1-phosphate (MTR-1-P) alone, however, were metabolized to two new products by these extracts. MTR kinase activity has previously been detected in these fruit extracts. These data indicate that MTR must be converted to MTR-1-P by MTR kinase before further metabolism can occur. The products of MTR-1-P metabolism were tentatively identified as α-keto-γ-methylthiobutyric acid (α-KMB) and α-hydroxy-γ-methylthiobutyric acid (α-HMB) by chromatography in several solvent systems. [³⁵S]α-KMB was found to be further metabolized to methionine and α-HMB by these extracts, whereas α-HMB was not. However, α-HMB inhibited the conversion of α-KMB to methionine. Both [U-¹⁴C]α-KMB and [U-¹⁴C]methionine, but not [U-¹⁴C]α-HMB, were converted to ethylene in tomato pericarp tissue. In addition, aminoethoxyvinylglycine inhibited the conversion of α-KMB to ethylene. These data suggest that the recycling pathway leading to ethylene is MTR → MTR-1-P → α-KMB → methionine → S-adenosylmethionine → 1-aminocyclopropane-1-carboxylic acid → ethylene.     

Cytochrome P-450-Dependent omega-Hydroxylation of Lauric Acid by Microsomes from Pea Seedlings

Microsomes from apical buds of pea (Pisum sativum L. var. Téléphone à rames) seedlings hydroxylate lauric acid at the ω-position. This oxidation is catalyzed by a cytochrome P-450 enzyme which differs from laurate hydroxylases previously described in microorganisms and mammals by its strict substrate specificity and the ability of low NADH concentrations to support unusually high oxidation rates. The apparent K_m for lauric acid was 20 micromolar. NADPH- and NADH-dependent laurate hydroxylation followed non-Michaelian kinetics with apparent K_m values ranging from 0.2 to 28 micromolar for NADPH, and 0.2 to 318 micromolar for NADH. When induced by the photomorphogenic photoreceptor phytochrome, the time course for the enhancement of laurate ω-hydroxylase was totally different from that of the cinnamic acid 4-hydroxylase, providing evidence for the existence of multiple cytochrome P-450 species in pea microsomes.     

Collagenolytic activity of rabbit V2-carcinoma growing at multiple sites.

Interaction between lanosterol and cytochrome P-450 purified from microsomes of anaerobically-grown Saccharomyces cerevisiae was studied. Lanosterol (4,4,14α-trimethyl-5α-cholesta-8,24-dien-3β-ol) stimulated the oxidation of NADPH by molecular oxygen in the presence of cytochrome P-450 and NADPH-cytochrome P-450 reductase both purified from S. cerevisiae microsomes. Lanosterol stimulated the reduction of cytochrome P-450 by NADPH with the cytochrome P-450 reductase, and induced Type I spectral change of cytochrome P-450. These observations suggest that lanosterol interacts to the substrate region of cytochrome P-450 of S. cerevisiae. Based on these facts, possible role of cytochrome P-450 in lanosterol metabolism in yeast cell is discussed.     

NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from Mortierella alpina

Fatty acid desaturases play an important role in maintaining the appropriate structure and function of biological membranes. The biochemical characterization of integral membrane desaturases, particularly ω3 and ω6 desaturases, has been limited by technical difficulties relating to the acquisition of large quantities of purified proteins, and by the fact that functional activities of these proteins were only tested in an NADH-initiated reaction system. The main aim of this study was to reconstitute an NADPH-dependent reaction system in vitro and investigate the kinetic properties of Mortierella alpina ω3 and ω6 desaturases in this system. After expression and purification of the soluble catalytic domain of NADPH–cytochrome P450 reductase, the NADPH-dependent fatty acid desaturation was reconstituted for the first time in a system containing NADPH, NADPH–cytochrome P450 reductase, cytochrome b5, M. alpina ω3 and ω6 desaturase and detergent. In this system, the maximum activity of ω3 and ω6 desaturase was 213.4 ± 9.0 nmol min⁻¹ mg⁻¹ and 10.0 ± 0.5 nmol min⁻¹ mg⁻¹, respectively. The highest k_cat/K_m value of ω3 and ω6 desaturase was 0.41 µM⁻¹ min⁻¹ and 0.09 µM⁻¹ min⁻¹ when using linoleoyl CoA (18:2 ω6) and oleoyl CoA (18:1 ω9) as substrates, respectively. M. alpina ω3 and ω6 desaturases were capable of using NADPH as reductant when mediated by NADPH–cytochrome P450 reductase; although, their efficiency is distinguishable from NADH-dependent desaturation. These results provide insights into the mechanisms underlying ω3 and ω6 fatty acid desaturation and may facilitate the production of important fatty acids in M. alpina.     

Studies on β-glucanases. Some properties of a bacterial endo-β-(1→3)-glucanase system

A commercial enzyme preparation, originally obtained from a Flavobacterium(Cytophaga), was fractionated by continuous electrophoresis, giving a protein fraction which hydrolysed laminarin, carboxymethylpachyman, barley β-glucan, lichenin and cellodextrin in random fashion. This enzymic activity was not very stable. Ion-exchange chromatography and molecular-sieve chromatography on Bio-Gel P-60 showed that this activity was due to two specific β-glucanases, an endo-β-(1→3)-glucanase and an endo-β-(1→4)-glucanase. The two enzymes occur in both high- and low-molecular-weight forms, the latter endo-β-(1→3)-glucanase having a molecular weight of about 16000.     

Beta-Amyloid Oligomers Activate Apoptotic BAK Pore for Cytochrome c Release

In Alzheimer’s disease, cytochrome c-dependent apoptosis is a crucial pathway in neuronal cell death. Although beta-amyloid (Aβ) oligomers are known to be the neurotoxins responsible for neuronal cell death, the underlying mechanisms remain largely elusive. Here, we report that the oligomeric form of synthetic Aβ of 42 amino acids elicits death of HT-22 cells. But, when expression of a bcl-2 family protein BAK is suppressed by siRNA, Aβ oligomer-induced cell death was reduced. Furthermore, significant reduction of cytochrome c release was observed with mitochondria isolated from BAK siRNA-treated HT-22 cells. Our in vitro experiments demonstrate that Aβ oligomers bind to BAK on the membrane and induce apoptotic BAK pores and cytochrome c release. Thus, the results suggest that Aβ oligomers function as apoptotic ligands and hijack the intrinsic apoptotic pathway to cause unintended neuronal cell death.     

Effects of Hormones on Changes in Cytochrome P-450, Prolyl Hydroxylase,and Glycerol Phosphate Acyltransferase in Primary Monolayer Cultures of Parenchymal Cells from Adult Rat Liver

Previous studies have shown that isolation and primary culture of rat hepatocytes in a standard, chemically defined medium is associated with selective changes in microsomal function. These changes were found to be selectively sensitive to addition of hormones to the culture medium. The concentration of cytochrome P-450 declined dramatically during the first 24 hours of incubation. However, cytochrome P₁-450, a form of the hemoprotein induced by polycyclic aromatic hydrocarbons, was resistant to this change. Cytochrome P₁-450 levels selectively rose during the first ten hours in culture and, thereafter, declined at a less rapid rate than did the cytochrome P-450 in normal hepatocytes or in cells prepared from phenobarbital pretreated animals. Addition of dexamethasone to the medium at the time of cell plating partially prevented the fall of cytochrome P-450 and of ¹⁴C-heme in microsomes prepared from hepatocytes derived from rats given 5¹⁴[C]-δ-aminolevulinic acid. This suggests that the steroid decreases degradation of the hemoprotein. As compared to the loss of cytochrome P-450 in cultures of normal hepatocytes, the hemoprotein fell to lower levels in hepatocytes prepared from regenerated liver four days after partial hepatectomy. This result may be related to the accelerated formation of the monolayer in the cultures of regenerated hepatocytes. Both sn-glycerol-3-phosphate acyltransferase activity and glycerol kinase activity declined in the first 24 hours of culture. The fall in the latter enzyme was partially prevented by addition of estradiol. Collagen prolyl hydroxylase, a newly discovered microsomal constituent of the hepatocyte, rose slightly during the first 24 hours in culture. This change was augmented threefold by addition of insulin to the medium. We conclude that the present hepatocyte culture system with its attendant changes in functional phenotype may be useful in better defining the role of hormones in modulating metabolic processes in the liver.     

Cloning and Functional Characterization of the Maize (Zea mays L.) Carotenoid Epsilon Hydroxylase Gene

The assignment of functions to genes in the carotenoid biosynthesis pathway is necessary to understand how the pathway is regulated and to obtain the basic information required for metabolic engineering. Few carotenoid ε-hydroxylases have been functionally characterized in plants although this would provide insight into the hydroxylation steps in the pathway. We therefore isolated mRNA from the endosperm of maize (Zea mays L., inbred line B73) and cloned a full-length cDNA encoding CYP97C19, a putative heme-containing carotenoid ε hydroxylase and member of the cytochrome P450 family. The corresponding CYP97C19 genomic locus on chromosome 1 was found to comprise a single-copy gene with nine introns. We expressed CYP97C19 cDNA under the control of the constitutive CaMV 35S promoter in the Arabidopsis thaliana lut1 knockout mutant, which lacks a functional CYP97C1 (LUT1) gene. The analysis of carotenoid levels and composition showed that lutein accumulated to high levels in the rosette leaves of the transgenic lines but not in the untransformed lut1 mutants. These results allowed the unambiguous functional annotation of maize CYP97C19 as an enzyme with strong zeinoxanthin ε-ring hydroxylation activity.     

Purification of Cytochrome P450 and Ferredoxin, Involved in Bisphenol A Degradation, from Sphingomonas sp. Strain AO1

In a previous study (M. Sasaki, J. Maki, K. Oshiman, Y. Matsumura, and T. Tsuchido, Biodegradation 16:449-459, 2005), the cytochrome P450 monooxygenase system was shown to be involved in bisphenol A (BPA) degradation by Sphingomonas sp. strain AO1. In the present investigation, we purified the components of this monooxygenase, cytochrome P450 (P450_bisd), ferredoxin (Fd_bisd), and ferredoxin reductase (Red_bisd). We demonstrated that P450_bisd and Fd_bisd are homodimeric proteins with molecular masses of 102.3 and 19.1 kDa, respectively, by gel filtration chromatography analysis. Spectroscopic analysis of Fd_bisd revealed the presence of a putidaredoxin-type [2Fe-2S] cluster. P450_bisd, in the presence of Fd_bisd, Red_bisd, and NADH, was able to convert BPA. The K_m and k_cat values for BPA degradation were 85 ± 4.7 μM and 3.9 ± 0.04 min⁻¹, respectively. NADPH, spinach ferredoxin, and spinach ferredoxin reductase resulted in weak monooxygenase activity. These results indicated that the electron transport system of P450_bisd might exhibit strict specificity. Two BPA degradation products of the P450_bisd system were detected by high-performance liquid chromatography analysis and were thought to be 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol based on mass spectrometry-mass spectrometry analysis. This is the first report demonstrating that the cytochrome P450 monooxygenase system in bacteria is involved in BPA degradation.