Ricinoleic Acid Catabolism in Peroxisomes

Peroxisomes from castor bean endosperm and mung bean hypocotyl completely degrade ricinoleic acid (12-D-hydroxy-9-cis-octadecenoic acid) to acetyl-CoA. Concomitant NADH formation occurred with a stoichiometry of 9 nmol NADH formed per 1 nmol ricinoleate degraded. At the C₈-intermediate level, where the hydroxy group of ricinoleic acid forms a barrier to β-oxidation, 2-hydroxyoctanoate and 2-oxooctanoate were detected as intermediates. 2-Hydroxyoctanoate was oxidized to 2-oxooctanoate with H₂O₂ producing a reaction exhibiting 1:1 stoichiometry of the products. The peroxisomes appeared to oxidize both isomers of racemic 2-hydroxyoctanoate. 2-Oxooctanoate was metabolized to heptanoyl-CoA (propionyl-CoA and acetyl-CoA) in a NAD-dependent, but ATP-independent, reaction. Heptanoate was not detected as an intermediate. Imidazole, an inhibitor of α-oxidation, did not effect the degradation of ricinoleate or 2-oxooctanoate. Arsenite, an inhibitor of oxidative decarboxylation, inhibited the metabolism of ricinoleate at the C₈-intermediate level, according to the accumulation of 2-oxooctanoate and the stoichiometry of concomitant NADH formation. Arsenite completely inhibited the metabolism of 2-oxooctanoate. It is concluded that the barrier caused by the hydroxy group of ricinoleic acid and prevention of β-oxidation at the C₈-intermediate level, is circumvented by an α-hydroxy acid oxidase reaction followed by an oxidative decarboxylation allowing return to the β-oxidation track.     

Catalytic reactions in the solar nebula: Implications for interstellar molecules and organic compounds in meteorites

Organic compounds in meteorites seem to have formed by Fischer-Tropsch-type, catalytic reactions of CO, H₂, and NH₃ in the solar nebula, at 360–400K and (4–10)×10^–6 atm. The onset of these reactions was triggered by the formation of catalytically active grains of magnetite and serpentine at these temperatures.Laboratory experiments show that the Fischer-Tropsch reaction gives a large kineticisotope fractionation of C¹²/C¹³, duplicating the hitherto unexplained fractionation in meteorites. All of the principal compound classes in meteorites are produced by this reaction, or a variant involving a brief excursion to higher temperatures. (1) normal, mono-, and dimethylalkanes (2)arenes andalkylarenes; (3) dimericisoprenoids from C₉ to C₁₄; (4)purines andpyrimidines, such as adenine, guanine, uracil, thymine, xanthine, etc.; (5)amino acids, including tyrosine and histidine; (6)porphyrin-like pigments; (7) aromaticpolymer with –OH and –COOH groups.These reactions may also have played a major role in the evolution of life: first, by converting carbon to a sufficiently non-volatile form to permit its accretion by the inner planets; second, by synthesizing organic compounds on the primitive planets whenever CO, H₂, NH₃, and clay or magnetite particles came together at the right temperature. Similar reactions in other solar nebulae may be the source of interstellar molecules, as first suggested by G. H. Herbig. Ten of the twelve polyatomic interstellar molecules have in fact been seen in these syntheses or in meteorites.This paper is a revised and abridged version of the author's article inScience 182 (1973), 781.     

FATTY ACIDS AND HYDROXY FATTY ACIDS IN THREE SPECIES OF FRESHWATER EUSTIGMATOPHYTES

The monocarboxylic fatty acids and hydroxy fatty acids of three species of freshwater microalgae—Vischeria punctata Vischer, Vischeria helvetica (Vischer et Pascher) Taylor, and Eustigmatos vischeri (Hulbert) Taylor, all from the class Eustigmatophyceae— were examined. Each species displayed a very similar distribution of fatty acids, the most abundant of which were 20:5n-3, 16:0, and 16:1n-7; C₁₈ polyunsaturated fatty acids were minor components. These fatty acid distributions closely resemble those found in marine eustigmatophytes but are quite distinct from those found in most other algal classes. These microalgae also contain long-chain saturated and unsaturated monohydroxy fatty acids. Two distinct types of hydroxy fatty acids were found: a series of saturated α-hydroxy acids ranging from C₂₄ to C₃₀ with a shorter series of monounsaturated α-hydroxy acids ranging from C₂₆ to C₃₀ together with a series of saturated β-hydroxy acids ranging from C₂₆ to C₃₀. The latter have not previously been reported in either marine or freshwater microalgae, although C₃₀ to C₃₄ midchain (ω-18)-hydroxy fatty acids have been identified in hydrolyzed extracts from marine eustigmatophytes of the genus Nannochloropsis, and C₂₂ to C₂₆ saturated and monounsaturated α-hydroxy fatty acids have been found in three marine chlorophytes. These findings have provided a more complete picture of the lipid distributions within this little studied group of microalgae as well as a range of unusual compounds that might prove useful chemotaxonomic markers. The functions of the hydroxy fatty acids are not known, but a link to the formation of the lipid precursors of highly aliphatic biopolymers is suggested.     

Tryptophan reaction with free radicals arisen from carbon tetrachloride in a model system. A mass spectrometric study

SUMMARY

The interaction between free radicals derived from the thermal decomposition of carbon tetrachloride and N-acetyl-d, l-tryptophan ethyl ester (TRPAE) under anaerobic and aerobic conditions was studied. The structure of the reaction products formed was deciphered by the GC/MS analysis of their trimethylsilyl derivatives. Under anaerobic conditions no formation of reaction products was detected. Under aerobic conditions the following products were identified:

• 1. A chloro hydroxy unsaturated adduct of TRPAE (2 isomers).

• 2. A dichloro hydroxy unsaturated adduct of TRPAE.

• 3. 12 products which are different pyrrolo[2,3-b]indol derivatives.

Some of the products appeared to have an hydroxyl group as a substituent, all of them contained chlorine and only one contained carbon from CCl₄. Interestingly, the formation of those adducts not containing CCl₃ would be missed during the regular procedures toxicologists use to determine the so-called ‘covalent binding’ employing ¹⁴CCl₄.

Concerning the potential relevance of these findings, we hypothesize that if interactions similar to those here reported occurred at least in part during CCl₄ poisoning, the resulting critical proteins containing tryptophan, e.g. membrane or other and enzymes containing the amino acid in their active center, might be impaired.     

Biodegradation of commercial linear alkyl benzenes byNocardia amarae

Laboratory degradation studies of two indigeneously produced linear alkyl benzenes byNocardia amarae MB-11 isolated from soil showed an overall degradation of linear alkyl benzenes isomers to the extent of 57–70%. Degradation of 2-phenyl isomers of linear alkyl benzenes was complete and faster than that of other phenyl position (C3–C7) isomers which were degraded to the extent of 40–72% only. Length of alkyl side chains (C₁₀–C₁₄) had little or no impact on the degradation pattern. Major metabolities detected were 2-, 3-and 4-phenyl butyric acids, phenyl acetic acid and cis, cis-muconic acid. Minor metabolites weretrans-cinnamic acid, 4-phenyl 3-butenoic acid and 3-phenyl pentanoic acid along with two unidentified hydroxy acids. On the basis of the formation pattern of these metabolities, three catabolic pathways of linear alkyl benzenes isomers inNocardia amarae MB-11 were postulated. All the phenyl position (C2–C7) isomers of C₁₀, C₁₂, and C₁₄ linear alkyl benzenes along with 3-phenyl and 5-phenyl isomers of C₁₁ and C₁₃ linear alkyl benzenes were degraded viacis,cis-muconic acid pathway. Other phenyl position isomers of C₁₁ and C₁₃ linear alkyl benzenes with phenyl substitution at even number carbon atoms were principally degraded via phenyl acetic acid pathway whiletrans-cinnamic acid formation provided a minor pathway     

Bacterial oxidation of cycloparaffinic hydrocarbons

A series of 10 branched-chain alkanes and 4 cycloalkanes were employed individually as elective culture substrates for bacteria in soil. Only 2-methylbutane and 2-methylpentane yielded bacteria, one each. Both bacteria grew at the expense of eachn-alkane from C₁ to C₂₂ but they were very selective for branched-chain substrates. Compounds with less branching were most readily utilized. Neither organism grew at the expense of various cycloalkanes as sole sources of carbon and energy. The 2-methylbutane isolate was studied in detail. Resting cell suspensions were able to produce α-ketoglutaric acid from each of the compounds the bacterium was able to utilize for growth. “Non-growth hydrocarbons” were also oxidized; in each case only neutral ketonic substances were detected. A series of cycloparaffins, from C₃- to C₈-membered rings, was oxidized to the corresponding cyclomonoketones. No oxidation products of cyclododecane (C₁₂), 1,4-cyclohexadiene (C₆) or benzene could be detected. The metabolic products identified are consistent with the formation of a cyclomonoalcohol as the immediate precursor of the ketone. The alcohol is formed from cycloalkanes, the cycloalkenes, and cycloalkene oxide as substrates. Alcohol formation from the first two probably takes place by independent parallel, rather than sequential, reaction pathways. The epoxide may be a non-obligate intermediate in the cyclomonoolefin conversion to the alcohol. Significant aspects of these conversions are discussed.     

Simulation of interstellar chemical evolution in a low temperature plasma

In order to simulate the interstellar chemical evolution, the chemical process was studied in a laboratory plasma flow. The apparatus was so designed as to establish the similarity between laboratory and cosmic conditions. The plasma temperature was found to be less than 100 K in the downstream region. HCN, HC₃N, H₂CO, and several kinds of hydrocarbons were produced from the plasma whose elementary composition was approximately same as the cosmic abundance. Based on the analysis by laser-induced-fluorescence method, HCN and HC₃N were concluded to be synthesized via CN loss reactions, while it was unlikely that the syntheses of C₂H₂ and H₂CO were related to the generation or depletion of C₂.     

Studies of the fischer-tropsch process on uranium catalysts

In the study of the catalytic reaction of uranium powder, phenylacetylene and dimethylacetylenedicarboxylate were converted to polymers via linear-oligomerization and cyclo-oligomerization reactions [6]. Our results indicate a new synthetic pathway of the carbon-carbon bond formation through the participation of f-orbital elements. In this report, reactions of methylenediiodide, diazomethane and ketene were studied. The products of these reactions, i.e., C₁ and C₄ alkanes and alkenes, suggested that the bond could be formed by the insertion of methylene radicals. An interesting phenomenon, where the carbon number of products increased with the duration of reaction period, was found.The role of the uranium element in the Fischer-Tropsch process was studied. Hydrogen and carbon monoxide were catalyzed to methane (60–70%), CH₃OH (20–25%), C₂H₅OH and CH₃OCH₂OCH₃ at one atmosphere pressure and 250 °C. The Fischer-Tropsch process could proceed through uranium alkoxide intermediates. This oxide-formation machanism was tested by the pyrolysis of uranium alkoxides.     

Regulation of oxaloacetate, aspartate, and malate formation in mesophyll protoplast extracts of three types of c(4) plants

The use of mesophyll protoplast extracts from various C₄ species has provided an effective method for studying light-and substrate-dependent formation of oxaloacetate, malate, and asparate at rates equivalent to whole leaf C₄ photosynthesis. Conditions regulating the formation of the C₄ acids were studied with protoplast extracts from Digitaria sanguinalis, an NADP-malic enzyme C₄ species, Eleusineindica, an NAD-malic enzyme C₄ species, and Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase C₄ species. Light-dependent induction of CO₂ fixation by the mesophyll extracts of all three species was relatively low without addition of exogenous substrates. Pyruvate, alanine and α-ketoglutarate, or 3-phosphoglycerate induced high rates of CO₂ fixation in the mesophyll extracts with oxaloacetate, malate, and aspartate being the primary products. In all three species, it appears that pyruvate, alanine, or 3-phosphoglycerate may serve as effective precursors to the formation of PEP for carboxylation through PEP-carboxylase in C₄ mesophyll cells. Induction by pyruvate or alanine and α-ketoglutarate was light-dependent, whereas 3-phosphoglycerate-induced CO₂ fixation was not.     

Transformation of steroids by Bacillus strains isolated from the foregut of water beetles (Coleoptera:Dytiscidae): I. Metabolism of androst-4-en-3,17-dione (AD)

Two Bacillus strains were isolated from the foregut of the water beetle Agabus affinis (Payk.) and tested for their steroid transforming ability. After incubation with androst-4-en-3,17-dione (AD), 13 different transformation products were detected. AD was hydroxylated at C₆, C₇, C₁₁ and C₁₄, resulting in formation of 6β-, 7α-, 11α- and 14α-hydroxy-AD. One strain also produced small amounts of 6β,14α-dihydroxy-AD. Partly, the 6β-hydroxy group was further oxidized to the corresponding 6-oxo steroids. In addition, a specific reduction of the Δ⁴-double bond was observed, leading to the formation of 5α-androstane derivatives. In minor yields the carbonyl functions at C₃ and C₁₇ were reduced leading to the formation of 3ξ-OH or 17β-OH steroids. EI mass spectra of the trimethylsilyl and O-methyloxime trimethylsilyl ether derivatives of some transformation products are presented for the first time.     

Fat metabolism in higher plants. LXII. The pathway of ricinoleic acid catabolism in the germinating castor bean (Ricinus communis L.) and pea (Pisum sativum L.)

With cell-free extracts from both germinating peas and castor beans, O-¹⁴Cricinoleate (¹⁴C located at odd-numbered positions in the carbon chain) was β-oxidized at least to the C₁₀ level. With the pea system, formation of unsaturated hydroxy acid intermediates to the C₁₂ level occurred. Acetyl-CoA was the primary product of β-oxidation activity. Although the pathway beyond the C₁₂-intermediate level was not resolved conclusively, two alternative routes may exist in the castor bean system to convert 4-hydroxy-decanoic acid to 2-keto-octanoate, one involving 4-keto-decanoate, the other 2-hydroxy-octanoate. Subsequent degradation of the 2-keto-octanoate tentatively involves an α-oxidation step, releasing CO₂ and heptanoic acid. Further β-oxidation of the latter is envisaged to yield propionyl and acetyl CoA. All necessary enzymes for the catabolism of ricinoleic acid to propionate appear to be associated with the cytosomes.     

Stereochemistry involved in the mechanism of action of dextransucrase in the synthesis of dextran and the formation of acceptor products

A proposed sequence of events in the synthesis of dextran and in the formation of acceptor products by dextransucrase from Leuconostoc mesenteroides B-512F has been developed with molecular models. The following mechanism is postulated: (1) two nucleophiles at the active site displace fructose from two sucrose molecules, giving two β-glucosyl intermediates; (2) these two β-glucosyl units rotate together so that the C₆-hydroxyl of each is apposed to the α-side of C₁ of the other; (3) one glucosyl unit assumes a boat conformation in which the bond to the enzyme is axial; (4) the C₆-hydroxyl oxygen of the other glucosyl unit makes a nucleophilic attack on C₁ of the first, displacing the enzyme nucleophile and making an α-1,6 bond; (5) rotations about the new α-1,6 linkage remove the transferred glucose from the active site. The free enzyme nucleophile attacks another sucrose as in step (1), and then steps (2)–(5) are repeated as the reducing-end glucosyl unit of the growing chain assumes the boat conformation and is attacked by the C₆-hydroxyl of the new glucosyl unit, which displaces the enzyme nucleophile and forms another α-1,6 linkage, about which rotations occur to remove the growing dextran chain from the active site. An additional feature of the mechanism presented here is a pair of enzymic proton-exchange groups, which protonate the glycosidic oxygen of sucrose to facilitate cleavage, and then remove a proton from the attacking C₆ hydroxyl during the polymerization reaction.Acceptors are polyhydroxy compounds which are capable of nucleophilic attack on enzyme-bound β-glucosyl or dextranosyl units to give α-glucosides or dextranosides. Noting the broad acceptor specificity of the enzyme and the unusual structure of some of the acceptor products, we have proposed that acceptor specificity is determined not by an enzymic binding site per se, but by the formation of hydrogen-bonded complexes between the acceptors and the glucosyl or dextranosyl enzyme intermediates. The acceptor attack on C₁ of the β-glucosyl enzyme is mediated by the same proton-exchange group as that proposed for catalysis of polymerization. It is shown that specific multiple hydrogen bonding to the glucosyl-enzyme intermediate can account for the formation of the observed acceptor products from α-methyl-d-glucoside, d-fructopyranose, isomaltose, maltose, β-d-mannopyranose, β-d-galactofuranose, cellobiose, lactose, β,β-trehalose, α,β-trehalose, and raffinose.     

Formation of electronically excited states during the interaction of p-benzoquinone with hydrogen peroxide

The reaction between p-benzoquinone and H₂O₂ in slightly alkaline solutions yields three major quinoid products that accumulate in the reaction mixture: (a) 2,3-epoxy-p-benzoquinone, (b) 2-hydroxy-p-benzoquinone and (c) p-benzohydroquinone. The reaction is accompanied by photoemission, probably originating from excited triplet 2-hydroxy-p-benzoquinone. These products originate from hydrogen peroxide and hydroxide nucleophilic addition to the C₂?C₃ double bond, as well as secondary redox interactions. The hydroxy substituent and the epoxide ring exert a substantial influence on the electronic distribution in the p-benzoquinone molecule leading to a decrease in the half-wave potential, as compared to the parent p-benzoquinone. The generation of electronically excited states is the result of reactions secondary to the nucleophilic additions involving 2-hydroxy-p-benzosemiquinone, H₂O₂ and hydroxyl radical. The process involves the primary oxidation of 2-hydroxy-p-benzosemiquinone by hydrogen peroxide, followed by oxidation of the semiquinone by hydroxyl radical leading to the formation of the electronically excited quinone. The decay of the excited triplet to the ground state is accompanied by photoemission with maximal intensity at 485–530 nm. Thermodynamic calculations along with an observed increase of photoemission intensity in anaerobiosis point to the triplet (n, π*) multiplicity of the excited state. The efficiency of chemiluminescence could be calculated as 10^?8 photons/2-hydroxy-p-benzoquinone molecule formed. Photoemission arising from the p-benzoquinone/H₂O₂ reaction was inhibited efficiently by addition of GSH to the reaction mixture. This may be due to deactivation of the triplet quinone by a 2-glutathionyl-p-benzohydroquinone adduct, involving thioether α-hydrogen atom-transfer to the triplet ketone.     

Effect of cultural conditions on the lipid profile of Thiobacillus ferroxidas

The intensitive investigations on the lipid profile of Thiobacillus ferrooxidans at various culture ages suggest some correlations of the lipid constitutents with the membrane-bound iron oxidation system. Phosphatidic acid, phosphatidyl serine and phosphatidyl ethanolamine were the major polar components; hydrocarbon, triglyceride and diglyceride were the main neutral components. Major fatty acids were C_16:0, C_16:1, C_16:3, C_18:1, C_18:3, C_22:1 while C_20:1, C_20:2, C_12:0, C_14:2, C_18:0, C_18:2, C_20:0, C_22:0 were found in trace amounts which also depended upon the phase of the growth. One lipoamino acid was identified as ornithine lipid in the polar fraction. Each and every component varied to some extent at different growth phasesindicating relationship of these lipids to the iron oxidation system of the strain.     

Epicuticular wax of Panicum virgatum

Leaf and stem wax of Panicum virgatum contains hydrocarbons (4%), esters (3%), free acids (2%), free alcohols (1%), triterpene alcohols (2%), β-diketones (69%) and hydroxy β-diketones (6%). Principal free alcohols range in chain length from C₂₆ to C₃₂. β-Diketones consist almost entirely of tritriacontane-12,14-dione and the hydroxy β-diketone consists only of 5(S)-5-hydroxytritriacontane-12,14-dione. The configuration of the hydroxyl group is the same as that of hydroxy β-diketones from festucoid grasses but opposite to that of the hydroxy β-diketone from Andropogon species.     

Photochemical reactions in interstellar grains photolysis of co,NH3, and H2O

A simulation of the organic layer accreted onto interstellar dust particles was prepared by slow deposition of a CO:NH₃:H₂O gas mixture on an Al block at 10K, with concomitant irradiation with vacuum UV. The residues were analyzed by GC-MS, HPLC, and near IR; a reaction pathway leading from NH₃ to complex alcohol, fatty acid, and amide products in 27 stages is postulated. The astronomical relevance and significance of the observations are discussed.     

Nuclear magnetic resonance studies of 5-aminolevulinate demonstrate multiple forms in aqueous solution

5-Aminolevulinic acid (ALA), the common precursor of heme and chlorophyll, can exist in a variety of forms at neutral pH. ¹³C NMR studies of [3-¹³C]ALA, [4-¹³C]ALA, and [5-¹³C]ALA have been used to demonstrate that the predominant species in solution under physiologic conditions is the ketone. The mole fraction of the hydrate is about 0.6%. To further substantiate the existence of the hydrate, ¹³C NMR was used to monitor ¹⁸O exchange at C₄ of [4-¹³C]ALA with H₂, ¹⁸O. Confirmation of the existence of the hydrate was achieved through direct observation by ¹H NMR. The mole fractions of the enol forms of ALA are each below 0.3%. Although direct observation of the enol forms of ALA has not been achieved, enol formation has been indirectly demonstrated by monitoring hydrogen exchange at the C₃ and C₅ methylene groups by ¹H NMR in D₂O. In neutral phosphate buffer, hydrogen exchange occurs readily at both C₃ and C₅ at a ratio of rates of 1:4. In N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid-KOH buffer the hydrogen exchange rates are more than an order of magnitude slower than in phosphate buffer, but the ratio of the exchange rates remains unchanged. The results suggest that phosphate catalyzes enolization at both C₃ and C₅. To evaluate the role of the C₅ substituent in the proton exchange reactions, levulinate and 5-chlorolevulinate (5-CLA) were also monitored for proton exchange at C₃ and C₅. For levulinate, the hydrogen exchange rates in phosphate buffer are two to three orders of magnitude slower than for ALA, and the rate of hydrogen exchange at C₅ is three times slower than hydrogen exchange at C₃. The enolization rate at C₅ of 5-CLA is identical to ALA while enolization at C₃ is about threefold slower for 5-CLA than ALA. These NMR and kinetic studies suggest that under physiologic conditions, ALA rapidly equilibrates between the ketone, the hydrate at C₄, and two or more different enols (C₃---C₄ and C₄---C₅). The alternative forms of ALA may be biologically significant as active site structures for ALA synthase, glutamate semialdehyde transaminase, or porphobilinogen synthase. These NMR studies have also elucidated the structures of condensation products of ALA which can be formed under physiologic conditions. The alternative forms of ALA, as well as the autocondensation products, may serve as the active toxin in porphyrias characterized by elevated ALA levels (e.g., lead poisoning).     

Analysis of the 220-MHz,P.M.R. spectra of some products of the amadori and heyns rearrangements

In order to establish whether p.m.r. spectroscopy is useful for identifying Amadori- and Heyns-rearrangement products, the p.m.r. spectra at 220 MHz of 16 rearrangement products derived from d-glucose or d-fructose and amino acids have been investigated. At pH 3, the protons of the NCH₂ group of N-substituted 1-amino-1-deoxy-d-fructose (Amadori-rearrangement products) resonate at δ 3.25–3.60 in D₂O and are shifted upfield by 0.3–0.6 p.p.m. at pH 9. These protons exchange with deuterium. Also, in D₂O there is an equilibrium of the acyclic, furanose, and pyranose structures, the last being favoured. At pH ? 7, the equilibrium is completely shifted to the β-pyranose form, which adopts exclusively the ²C₅ conformation. At pH 3, the equilibrium favours the β-furanose form. At pH 3, H-1e and H-1a of N-substituted 2-amino-2-deoxy-d-glucoses (Heyns-rearrangement products) resonate at δ 5.55 and 5.04, respectively. At pH 9, the signal for H-2 is shifted upfield by 0.2–0.7 p.p.m. In D₂O solution, these compounds exist as an equilibrium of α- and β-pyranose forms in the ⁴C₁ conformation. The α anomer is stabilised by the amino acid group at position 2. At pH 3, the αβ-ratio is 2–4:1, and, at pH 9, 1.0–1.1:1.     

Discovery of alkenones with variable methylene‐interrupted double bonds: implications for the biosynthetic pathway

Alkenones (C₃₇–C₄₀) are highly specific biomarkers produced by certain haptophyte algae in ocean and lacustrine environments and have been widely used for paleoclimate studies. Unusual shorter‐chain alkenones (SCA; e.g., C₃₅ and C₃₆) have been found in environmental and culture samples, but the origin and structure of these compounds are much less understood. The marine alkenone producer, Emiliania huxleyi CCMP2758 strain, was reported with abundant C_35:2Me (?^{12, 19}) alkenones when cultured at 15°C (Prahl et al. 2006). Here we show, when this strain is cultured at 4°C–10°C, that CCMP2758 produces abundant C_35:3Me, C_36:3Me, and small amounts of C_36:3Et alkenones with unusual double‐bond positions of ?^{7, 12, 19}. We determine the double‐bond positions of the C_35:3Me and C_36:3Me alkenones by GC‐MS analysis of the dimethyl disulfide and cyclobutylamine derivatives, and we provide the first temperature calibrations based on the unsaturation ratios of the C₃₅ and C₃₆ alkenones. Previous studies have found C_35:2Me (?^{14, 19}) and C_36:2Et (?^{14, 19}) alkenones with three‐methylene interruption in the Black Sea sediments, but this is the first reported instance of alkenones with a mixed three‐ and five‐methylene interruption configuration in the double‐bond positions. The discovery of these alkenones allows us to propose a novel biosynthetic scheme, termed the SCA biosynthesis pathway, that simultaneously rationalizes the formation of both the C_35:3Me (?^{7, 12, 19}) alkenone in our culture and the ?^{14, 19} Black Sea type alkenones without invoking new desaturases for the unusual double‐bond positions.     

Co-function of C3-and C4-photosynthetic pathways in C3, C4 and C3-C4 intermediate Flaveria species

The potential for C₄ photosynthesis was investigated in five C₃-C₄ intermediate species, one C₃ species, and one C₄ species in the genus Flaveria, using ¹⁴CO₂ pulse-¹²CO₂ chase techniques and quantum-yield measurements. All five intermediate species were capable of incorporating ¹⁴CO₂ into the C₄ acids malate and aspartate, following an 8-s pulse. The proportion of ¹⁴C label in these C₄ products ranged from 50–55% to 20–26% in the C₃-C₄ intermediates F. floridana Johnston and F. linearis Lag. respectively. All of the intermediate species incorporated as much, or more, ¹⁴CO₂ into aspartate as into malate. Generally, about 5–15% of the initial label in these species appeared as other organic acids. There was variation in the capacity for C₄ photosynthesis among the intermediate species based on the apparent rate of conversion of ¹⁴C label from the C₄ cycle to the C₃ cycle. In intermediate species such as F. pubescens Rydb., F. ramosissima Klatt., and F. floridana we observed a substantial decrease in label of C₄-cycle products and an increase in percentage label in C₃-cycle products during chase periods with ¹²CO₂, although the rate of change was slower than in the C₄ species, F. palmeri. In these C₃-C₄ intermediates both sucrose and fumarate were predominant products after a 20-min chase period. In the C₃-C₄ intermediates, F. anomala Robinson and f. linearis we observed no significant decrease in the label of C₄-cycle products during a 3-min chase period and a slow turnover during a 20-min chase, indicating a lower level of functional integration between the C₄ and C₃ cycles in these species, relative to the other intermediates. Although F. cronquistii Powell was previously identified as a C₃ species, 7–18% of the initial label was in malate+aspartate. However, only 40–50% of this label was in the C-4 position, indicating C₄-acid formation as secondary products of photosynthesis in F. cronquistii. In 21% O₂, the absorbed quantum yields for CO₂ uptake (in mol CO₂·[mol quanta]^-1) averaged 0.053 in F. cronquistii (C₃), 0.051 in F. trinervia (Spreng.) Mohr (C₄), 0.052 in F. ramosissima (C₃-C₄), 0.051 in F. anomala (C₃-C₄), 0.050 in F. linearis (C₃-C₄), 0.046 in F. floridana (C₃-C₄), and 0.044 in F. pubescens (C₃-C₄). In 2% O₂ an enhancement of the quantum yield was observed in all of the C₃-C₄ intermediate species, ranging from 21% in F. ramosissima to 43% in F. pubescens. In all intermediates the quantum yields in 2% O₂ were intermediate in value to the C₃ and C₄ species, indicating a co-function of the C₃ and C₄ cycles in CO₂ assimilation. The low quantum-yield values for F. pubescens and F. floridana in 21% O₂ presumably reflect an ineffcient transfer of carbon from the C₄ to the C₃ cycle. The response of the quantum yield to four increasing O₂ concentrations (2–35%) showed lower levels of O₂ inhibition in the C₃-C₄ intermediate F. ramosissima, relative to the C₃ species. This indicates that the co-function of the C₃ and C₄ cycles in this intermediate species leads to an increased CO₂ concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase and a concomitant decrease in the competitive inhibition by O₂.Abbreviations PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - RuBP ribulose-1,5-bisphosphate