Abscisic Acid Biosynthesis in Leaves and Roots of Xanthium strumarium

Research on the biosynthesis of abscisic acid (ABA) has focused primarily on two pathways: (a) the direct pathway from farnesyl pyrophosphate, and (b) the indirect pathway involving a carotenoid precursor. We have investigated which biosynthetic pathway is operating in turgid and stressed Xanthium leaves, and in stressed Xanthium roots using long-term incubations in ¹⁸O₂. It was found that in stressed leaves three atoms of ¹⁸O from ¹⁸O₂ are incorporated into the ABA molecule, and that the amount of ¹⁸O incorporated increases with time. One ¹⁸O atom is incorporated rapidly into the carboxyl group of ABA, whereas the other two atoms are very slowly incorporated into the ring oxygens. The fourth oxygen atom in the carboxyl group of ABA is derived from water. ABA from stressed roots of Xanthium incubated in ¹⁸O₂ shows a labeling pattern similar to that of ABA in stressed leaves, but with incorporation of more ¹⁸O into the tertiary hydroxyl group at C-1′ after 6 and 12 hours than found in ABA from stressed leaves. It is proposed that the precursors to stress-induced ABA are xanthophylls, and that a xanthophyll lacking an oxygen function at C-6 (carotenoid numbering scheme) plays a crucial role in ABA biosynthesis in Xanthium roots. In turgid Xanthium leaves, ¹⁸O is incorporated into ABA to a much lesser extent than it is in stressed leaves, whereas exogenously applied ¹⁴C-ABA is completely catabolized within 48 hours. This suggests that ABA in turgid leaves is either (a) made via a biosynthetic pathway which is different from the one in stressed leaves, or (b) has a half-life on the order of days as compared with a half-life of 15.5 hours in water-stressed Xanthium leaves. Phaseic acid showed a labeling pattern similar to that of ABA, but with an additional ¹⁸O incorporated during 8′-hydroxylation of ABA to phaseic acid.     

An evidence of the peroxidase-dependent oxygen transfer from hydrogen peroxide to sulfides

Horseradish- and chloro-peroxidase catalyzed oxidation of sulfides have been investigated. Thioanisoles were oxygenated to the corresponding sulfoxides by such peroxidases at the expense of H2O2. Dealkylation was observed only in the chloroperoxidase-dependent oxidations of p-methoxy- and p-iso-propoxy-thioanisoles. The experiments with 18O-labeled H2O2 indicated that an oxygen atom of H2O2 is incorporated into the sulfoxides. These research lead to the conclusion that compound I or II is capable of acting as an oxygen donor as well as an electron acceptor.     

Incorporation of oxygen into abscisic Acid and phaseic Acid from molecular oxygen

Abscisic acid accumulates in detached, wilted leaves of Xanthium strumarium. When these leaves are subsequently rehydrated, phaseic acid, a catabolite of abscisic acid, accumulates. Analysis by gas chromatography-mass spectrometry of phaseic acid isolated from stressed and subsequently rehydrated leaves placed in an atmosphere containing 20% ¹⁸O₂ and 80% N₂ indicates that one atom of ¹⁸O is incorporated in the 6′-hydroxymethyl group of phaseic acid. This suggests that the enzyme that converts abscisic acid to phaseic acid is an oxygenase.

Analysis by gas chromatography-mass spectrometry of abscisic acid isolated from stressed leaves kept in an atmosphere containing ¹⁸O₂ indicates that one atom of ¹⁸O is present in the carboxyl group of abscisic acid. Thus, when abscisic acid accumulates in water-stressed leaves, only one of the four oxygens present in the abscisic acid molecule is derived from molecular oxygen. This suggests that either (a) the oxygen present in the 1′-, 4′-, and one of the two oxygens at the 1-position of abscisic acid arise from water, or (b) there exists a stored precursor with oxygen atoms already present in the 1′- and 4′-positions of abscisic acid which is converted to abscisic acid under conditions of water stress.

     

On the mechanism of action of xanthine oxidase. Evidence in support of an oxo transfer mechanism in the molybdenum-containing hydroxylases

The mechanism of action of xanthine oxidase has been investigated using single-turnover experiments in an effort to determine the primary source of the oxygen atom incorporated into product in the course of catalysis. It is found from mass spectroscopic analysis of the uric acid generated in these experiments that when 16O-labeled enzyme in [18O]H2O is reacted with substoichiometric amounts of xanthine (under conditions where no enzyme molecule is likely to react with more than one substrate molecule), the uric acid isolated from the reaction mixture contains 16O at position 8 of the purine ring. Conversely, when 18O-labeled enzyme in [16O]H2O is exposed to substoichiometric xanthine, 18O is incorporated into the product uric acid. These results strongly support a variety of chemical studies with model molybdenum complexes suggesting that the oxygen atom of the Mo = O group known to be present at the active site of xanthine oxidase is transferred to product in the course of catalysis. The mechanistic implications of the present work are discussed.     

Plant sulfite oxidase as novel producer of H2O2: combination of enzyme catalysis with a subsequent non-enzymatic reaction step

Sulfite oxidase (EC 1.8.3.1) from the plant Arabidopsis thaliana is the smallest eukaryotic molybdenum enzyme consisting of a molybdenum cofactor-binding domain but lacking the heme domain that is known from vertebrate sulfite oxidase. While vertebrate sulfite oxidase is a mitochondrial enzyme with cytochrome c as the physiological electron acceptor, plant sulfite oxidase is localized in peroxisomes and does not react with cytochrome c. Here we describe results that identified oxygen as the terminal electron acceptor for plant sulfite oxidase and hydrogen peroxide as the product of this reaction in addition to sulfate. The latter finding might explain the peroxisomal localization of plant sulfite oxidase. 18O labeling experiments and the use of catalase provided evidence that plant sulfite oxidase combines its catalytic reaction with a subsequent non-enzymatic step where its reaction product hydrogen peroxide oxidizes another molecule of sulfite. In vitro, for each catalytic cycle plant SO will bring about the oxidation of two molecules of sulfite by one molecule of oxygen. In the plant, sulfite oxidase could be responsible for removing sulfite as a toxic metabolite, which might represent a means to protect the cell against excess of sulfite derived from SO2 gas in the atmosphere (acid rain) or during the decomposition of sulfur-containing amino acids. Finally we present a model for the metabolic interaction between sulfite and catalase in the peroxisome.     

Three hydroxylations incorporating molecular oxygen in the aerobic biosynthesis of ubiquinone in Escherichia coli.

The biosynthetic origin of the oxygen atoms of ubiquinone 8 from aerobically grown Escherichia coli was studied by 18O labeling. An apparatus was developed which allowed the growth of cells under a defined atmosphere. Mass spectral analysis of ubiquinone 8 from cells grown under highly enriched 18O2 showed that three oxygen atoms of the quinone are derived from molecular oxygen. It was established that the molecular oxygen is incorporated into the two methoxyl groups (at C-5 and C-6) and one of the carbonyl positions of the ubiquinone molecule by demonstrating that only one of the incorporated oxygens will exchange with water under acidic conditions that specifically catalyze the exchange of carbonyl, but not methoxyl, oxygens. That the C-4 carbonyl oxygen is derived from molecular oxygen was shown by the incorporation of three atoms of 18O2 into ubiquinone 8 biosynthesized from added 4-hydroxybenzoic acid. Comparison of ubiquinone 8 and menaquinone 8 from E. coli grown under 18O2 confirmed that the labeled carbonyl oxygen of the [18O2]ubiquinone 8 is incorporated biosynthetically and not by chemical exchange in the cell. It is concluded that the three hydroxylation reactions involved in the pathway for the aerobic biosynthesis of ubiquinone are all catalyzed by monooxygenases. The implications of this study for the anaerobic biosynthesis of ubiquinone 8 in E coli are discussed.     

Oxidation of Nicotinic Acid by a Bacillus Species: Purification and Properties of Nicotinic Acid and 6-Hydroxynicotinic Acid Hydroxylases

The enzymes of a Bacillus species that hydroxylate nicotinic acid to 6-hydroxynicotinic acid and 6-hydroxynicotinic acid to 2,6-dihydroxynicotinic acid were purified and characterized. The purified enzymes contained approximately two molecules of flavine and eight molecules of iron per molecule of enzyme. The enzymes were large (molecular weight, 400,000 to 450,000) and appeared to consist of subunits.     

Dioxygen transfer during vitamin K dependent carboxylase catalysis.

The vitamin K dependent carboxylase of liver microsomes is involved in the posttranslational modification of certain serine protease zymogens which are critical components of the blood clotting cascade. During coupled carboxylation/oxygenation this carboxylase converts glutamate residues, dihydrovitamin K, CO2, and O2 to a gamma-carboxyglutamyl (Gla) residue, vitamin K (2R,3S)-epoxide, and H2O with a stoichiometry of 1:1 for all substrates and products. In this paper we investigate the role of molecular oxygen in the reaction by following the course of the oxygen atoms using 18O2. Two different mass spectroscopic techniques, electron ionization positive ion mass spectrometry and supercritical fluid chromatography-negative ion chemical ionization mass spectrometry, were used to quantitate the amount of 18O incorporation into the various oxygens of the vitamin K epoxide product. We found that 0.95 mol atoms of oxygen were incorporated into the epoxide oxygen, 0.05 mol atoms of oxygen were incorporated into the quinone oxygen of vitamin K epoxide, and the remaining ca. 1.0 mol atoms of oxygen were incorporated into H2O. No incorporation of oxygen into vitamin K epoxide from 50% H2(18)O was observed. Thus, the carboxylase operates as a dioxygenase 5% of the time during carboxylation/oxygenation. The relevance of these findings with respect to the nonenzymic "basicity enhancement" model proposed by Ham and Dowd [(1990) J. Am. Chem. Soc. 112, 1660-1661] is discussed.     

Evidence for a universal pathway of abscisic Acid biosynthesis in higher plants from o incorporation patterns

Previous labeling studies of abscisic acid (ABA) with ¹⁸O₂ have been mainly conducted with water-stressed leaves. In this study, ¹⁸O incorporation into ABA of stressed leaves of various species was compared with ¹⁸O labeling of ABA of turgid leaves and of fruit tissue in different stages of ripening. In stressed leaves of all six species investigated, avocado (Persea americana), barley (Hordeum vulgare), bean (Phaseolus vulgaris), cocklebur (Xanthium strumarium), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum), ¹⁸O was most abundant in the carboxyl group, whereas incorporation of a second and third ¹⁸O in the oxygen atoms on the ring of ABA was much less prominent after 24 h in ¹⁸O₂. ABA from turgid bean leaves showed significant ¹⁸O incorporation, again with highest ¹⁸O enrichment in the carboxyl group. The ¹⁸O-labeling pattern of ABA from unripe avocado mesocarp was similar to that of stressed leaves, but in ripe fruits there was, besides high ¹⁸O enrichment in the carboxyl group, also much additional ¹⁸O incorporation in the ring. In ripening apple fruit tissue (Malus domestica), singly labeled ABA was most abundant with more ¹⁸O incorporated in the tertiary hydroxyl group than in the carboxyl group of ABA. Smaller quantities of this monolabeled product (C-1′-¹⁸OH) were also detected in the stressed leaves of barley, bean, and tobacco, and in avocado fruits. It is postulated that a large precursor molecule yields an aldehyde cleavage product that is, in some tissues, rapidly converted to ABA with retention of ¹⁸O in the carboxyl group, whereas in ripening fruits and in the stressed leaves of some species the biosynthesis of ABA occurs at a slower rate, allowing this intermediate to exchange ¹⁸O with water. On the basis of ¹⁸O-labeling patterns observed in ABA from different tissues it is concluded that, despite variations in precursor pool sizes and intermediate turnover rates, there is a universal pathway of ABA biosynthesis in higher plants which involves cleavage of a larger precursor molecule, presumably an oxygenated carotenoid.     

Spontaneous Hydrolysis and Dehydration of Dehydroascorbic Acid in Aqueous Solution

The interaction of water with dehydroascorbic acid was examined by incubating dehydroascorbic acid and ascorbic acid in¹⁸O-labeled water for various amounts of time and then oxidizing the products with hydrogen peroxide or reducing the products with mercaptoethanol, with analysis by gas chromatography mass spectrometry. Based on mass changes, dehydroascorbic acid readily exchanged three oxygen atoms with H₂¹⁸O. When mercaptoethanol was used to reduce dehydroascorbic acid (which had been incubated in H₂¹⁸O) to ascorbic acid, the newly formed ascorbic acid also contained three labeled oxygen atoms. However, ascorbic acid incubated in H₂¹⁸O for the same amount of time under identical conditions exchanged only two labeled oxygen atoms. Electron impact mass spectrometry of derivatized ascorbic acid created a decarboxylation product which had only two labeled oxygen atoms, regardless if 3-oxygen-labeled or 2-oxygen-labeled ascorbic acid was the parent compound, isolating the extra oxygen addition to carbon 1. These data suggest that dehydroascorbic acid spontaneously hydrolyzes and dehydrates in aqueous solution and that the hydrolytic-hydroxyl oxygen is accepted by carbon 1. Ascorbic acid, on the other hand, does not show this same tendency to hydrolyze.     

A study of the mechanism of quercetin oxygenation by 18O labelling. A comparison of the mechanism with that of haem degradation.

The mechanism of quercetin oxygenation, which is formally analogous to haem degradation, was studied by using 18O labelling. In both the enzymic oxygenation (catalysed by quercetinase) and the non-enzymic reaction (base-catalysed), both oxygen atoms incorporated into product were derived from a single oxygen molecule. Quercetin oxygenation therefore occurs by a classical dioxygenase mechanism and is not an appropriate model for study of the mechanism of haem catabolism.     

Incorporation of 18 O into homovanillic acid of rat brain during exposure to oxygen 18 containing atmospheres

Rats were exposed to air containing ¹⁸O₂ at atmospheric pressure. $\text{In vivo}$ incorporation of ¹⁸O in brain homovanillic acid (HVA) was determined by gas chromatography-mass spectrometry. One ¹⁸O atom was incorporated into each molecule of HVA indicating that tyrosine is the predominant precursor of brain dopamine and that the oxygen in the 3-position is of atmospheric origin. Intraperitoneal administration of ¹⁸O-enriched water did not alter the ¹⁸O content of brain HVA Mass fragmentography (2) was used to measure the increase in ¹⁸O and the decrease in ¹⁶O in HVA from rat brain over several hours of exposure to an ¹⁸O enriched atmosphere. These experiments demonstrate the possibility to pulse label brain dopamine and its metabolites by $\text{in vivo}$ inhalation of stable oxygen isotopes. The procedure should be useful for quantitative determinations of the turnover of brain dopamine in animals and man.     

The mechanism of conversion of rat liver xanthine dehydrogenase from an NAD+-dependent form (type D) to an O2-dependent form (type O).

Rat liver xanthine dehydrogenase, type D, has been isolated directly from crude extracts as an antibody complex and its properties compared with those of two oxidase forms of the enzyme, heat-treated type O and trypsin-treated type O, also isolated as antibody complexes. The type D antibody complex displays electron acceptor specificities and electron paramagnetic resonance properties characteristic of an NAD⁺-dependent dehydrogenase whereas the trypsin-treated type O complex behaves as an O₂-utilizing oxidase. The heat-treated type O complex displays intermediate behavior. After electrophoresis in dodecyl sulfate-urea-acrylamide gels, type D and heated type O enzymes show single polypeptide bands, each of approximately 150,000 molecular weight. The trypsinized type O also shows one major band but with an approximate molecular weight of 130,000. Purified type D enzyme, when proteolytically treated, is converted to an oxidase with increased mobility on polyacrylamide gels. The 150,000 molecular weight subunit is cleaved into smaller subunits during proteolysis. Treatment with 5,5′-dithiobis-(2-nitrobenzoic acid) converts the type D enzyme, whether isolated as the purified enzyme or as the immune precipitate, to type O enzyme in a time-dependent manner. Titration of type D and the two type O antibody complexes with 5,5′-dithiobis-(2-nitrobenzoic acid) reveals that type D and heated type O each has approximately 28 reactive sulfhydryls, whereas the trypsinized type O has only 8 such groups. Many of the free sulfhydryls are vicinal and form disulfide bonds during the conversion to an oxidase by this reagent. Unproteolyzed preparations of type O rat liver enzyme and milk xanthine oxidase are converted to type D enzymes by treatment with dithiothreitol. The converted enzymes display electron acceptor specificities and epr properties characteristic of an NAD⁺-dependent dehydrogenase molecule.     

Early biosynthetic pathway to abscisic acid in Cercospora cruenta

A new biosynthetic intermediate of ABA, (2Z,4E)-gamma-ionylideneacetaldehyde, was isolated from young mycelia of Cercospora cruenta. Under an (18)O2 atmosphere, an oxygen atom of this endogenous aldehyde was exclusively labeled. Similarly, three (18)O atoms were incorporated into the ABA molecule recovered after prolonged incubation; selectively labeled were one of the carboxyl oxygen atoms and the two on the ring portion of ABA. A feeding experiment with [1-(13)C]glucose proved the exclusive operation of the mevalonate pathway for the formation of both ABA and beta-carotene. These results suggest that (2Z,4E)-gamma-ionylideneacetaldehyde can be a key ABA biosynthetic intermediate formed by the oxidative cleavage of a carotenoid precursor.     

Trichothecene Biosynthesis in Fusarium sporotrichioides: Origin of the Oxygen Atoms of T-2 Toxin

Mass spectral analysis of T-2 toxin formed during the growth of Fusarium sporotrichioides (ATCC 24043) in the presence of H₂¹⁸O showed incorporation of up to three ¹⁸O atoms per toxin molecule. The carbonyl oxygens of the acetates at C-4 and C-15 and of the isovalerate at C-8 were derived from H₂O. Toxin formed in the presence of ¹⁸O molecular oxygen incorporated up to six ¹⁸O atoms per toxin molecule. The overall incorporation was 78 and 92% of toxin molecules labeled for H₂¹⁸O and ¹⁸O₂ labeled samples, respectively. The oxygens of position 1, the 12,13-epoxide, and the hydroxyl groups at C-3, C-4, C-8, and C-15 were all derived from molecular oxygen.     

Origin of the chlorophyll b formyl oxygen in Chlorella vulgaris.

Chlorophyll (Chl) b is an accessory light-harvesting pigment of plants and chlorophyte algae. Chl b differs from Chl a in that the 3-methyl group on ring B of chl a is replaced by a 3-formyl group on Chl b. The present study determined the biosynthetic origin of the Chl b formyl oxygen in in vivo labeling experiments. A mutant strain of the unicellular chlorophyte Chlorella vulgaris, which can not synthesize Chls when cultured in the dark but rapidly greens when transferred to the light, was grown in the dark for several generations to deplete Chls, and then the cells were transferred to the light and allowed to form Chls in a controlled atmosphere containing 18O2. Chl a and Chl b were purified from the cells and analyzed by high-resolution mass spectroscopy. Analysis of the mass spectra indicated that over 76% of the Chl a molecules had incorporated an atom of 18O. For Chl b, 58% of the molecules had incorporated an atom of 18O at one position and 34% of the molecules had incorporated an atom of 18O at a second position. These results demonstrate that the isocyclic ring keto oxygen of both Chl a and Chl b, as well as the formyl oxygen of Chl b, is derived from O2.     

Involvement of a single thiol group in the conversion of the NAD+-dependent activity of rat liver xanthine oxidoreductase to the O2-dependent activity.

The effects of 2-iodosobenzoic acid, 4-chloromercuribenzoate, 5,5'-dithiobis-(2-nitrobenzoic acid) and tetraethylthioperoxydicarbonic diamide (disulphiram) on the NAD+-dependent activity of xanthine oxidoreductase from rat liver were investigated. Only disulphiram converted the NAD+-dependent activity into the O2-dependent activity quantitatively, without changing the xanthine hydroxylation rate. The modification process was a first-order reaction with respect to time (min) and disulphiram concentration (microM). The kinetic data showed that modification of single thiol group is sufficient for loss of the enzymic activity towards NAD+ as electron acceptor. The complete protection afforded by NAD+ against the action of disulphiram suggests that the essential thiol group may be involved in binding of NAD+ to the xanthine oxidoreductase molecule.     

A likely molecular basis of the susceptibility of Giardia lamblia towards oxygen

Giardia lamblia is an amitochondrial protozoan susceptible to oxygen, but the molecular basis for it remains unclear. A Giardia NAD(P)H:menadione oxidoreductase (DT-diaphorase) is known to catalyse a single electron transfer reaction with quinones as the likely two-electron acceptor when oxygen is absent. Here we overexpressed this enzyme in Giardia trophozoites and observed a significantly enhanced susceptibility of the cells towards oxygen. A knock-down of this enzyme resulted, however, in more oxygen-tolerant Giardia cells growing equally well under anaerobic and aerobic conditions. The function of DT-diaphorase could be thus a major, if not the only, cause for the oxygen susceptibility of Giardia. Overexpressed DT-diaphorase is accompanied by increased intracellular hydrogen peroxide. An overexpression of Fe-superoxide dismutase in Giardia led also to a similarly heightened sensitivity to oxygen. Thus, generation of H2O2 from superoxide anion likely produced from DT-diaphorase catalysed reaction using oxygen as electron acceptor may constitute the molecular basis for Giardia susceptibility to oxygen. A functional homologue of DT-diaphorase in Giardia, NADH oxidase, uses oxygen as the preferred electron acceptor and reduces it to water. Overexpression of this enzyme in Giardia resulted in significantly enhanced growth under aerobic conditions. Giardia NADH oxidase could be thus an instrumental enzyme for the organism to adapt to and to tolerate an aerobic living environment.     

L-氨基酸氧化酶的结构、底物谱、家族进化及应用研究进展

L-氨基酸氧化酶(LAAO)是一类生物体内参与氨基酸氧化代谢的重要氧化还原酶,能够以氧分子为电子受体催化L-氨基酸氧化脱氨,生成相应的酮酸、氨(NH₃)和过氧化氢(H₂O₂).近期发现有些LAAO能够专一性识别特定氨基酸,而不受其他种类氨基酸的干扰,因而在手性胺类化合物拆分、α-酮酸生物合成、临床样本、食品及氨基酸发酵过程中氨基酸含量检测等领域都发挥着重要作用.本文重点综述目前研究报道的底物专一性LAAO,总结并比较这些酶的酶学性质、结构功能,以及家族进化规律等,并进一步讨论这些酶在生物催化及氨基酸检测中的应用.本综述将为底物特异性LAAO的分子机制研究及产业应用研究提供重要的素材和指导.     

The living state and cancer

Most inanimate systems are build of closed-shell molecules in which electrons lack excitability and mobility. These electrons can be rendered reactive and mobile by taking out some of them, desaturating the system electronically. Single electrons can be taken out of molecules by transfer to an external acceptor, creating two radicals that form a biradical having no net charge. The living state is such an electronically desaturated state. The universal electron acceptor of the biosphere is oxygen. Before light and O2 appeared, a weak electron acceptor could occur through linkage of two C=O groups to glyoxal and addition of a methyl group. The resulting methylglyoxal, being a weak acceptor, could lead to only a low degree of desaturation and thus to formation of only the simple life forms extant during this dark and anaerobic period--the alpha period. During the subsequent aerobic beta period, more highly differentiated life forms could develop because of occurrence of O2, a strong electron acceptor leading to a greater degree of desaturation. When dividing, however, beta-type cells return partially to the proliferative alpha state. The process of electron (charge) transfer, described here in two models, depends on the dielectric constant of the medium and the relative concentration of SH and methylglyoxal. Structure-building proteins that perform the main biological functions carry with them this chemical mechanism of their desaturation. Central to the mechanism is the NH2 of lysine that attaches a methylglyoxal. Through folding of the side chain, the CO groups of resulting Schiff bases can come in touch with the NH's of the peptide chain and accept electrons from it, desaturating it. Ascorbic acid is the catalyst of this charge transfer, which brings protein into the living state. Purified protein is inanimate matter. Manganese and oxygen form part of the chemical mechanism of desaturation, and the charge transfer reactions studied were found to be autocatalytic. It follows from the above observations that a cancer cell is a cell trapped in the alpha state.