Biosynthesis of a hydroxy fatty acid polymer, cutin. Identification and biosynthesis of 16-oxo-9- or 10-hydroxypalmitic acid, a novel compound in Vicia faba

Pe, Kolattukudy

doi:10.1021/bi00704a008

Cited by 27 publications

(19 citation statements)

References 15 publications

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“…The intense ion at m/e 275 represented the co-hydroxy end of the 10,16-dihydroxy C16 acid of cutin and the carboxyl end gave a rise to the ion at m/e 319 containing 2 deuterium atoms (incorporated during the reduction of the carboxyl group by LiAlD4). The ion at m/e 290 contained 1 deuterium atom which was incorporated during the LiAlD4 reduction of the woxo group of 16-oxo-9-hydroxy C16 acid as discussed elsewhere (13). The observation that the ion at m/e 303 (in the spectrum of hydrogenolysate) has been replaced by an ion at m/e 305 is in accordance with the conclusions that it originated from the carboxyl end of the w-oxo acid.…”

Section: Effect Of Enzyme Concentration and Time On Hydrolysis Ofsupporting

confidence: 79%

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Production of a Novel Extracellular Cutinase by the Pollen and the Chemical Composition and Ultrastructure of the Stigma Cuticle of Nasturtium (Tropaeolum majus)

1977

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Germinating nasturtium polen (Tropaeolum majus) is shown to excrete an enzyme(s) which hydrolyzes all types of monomers from biosyntheticafly labeled cutin andp-nitrophenyl esters, which are model substrates for fungal cutases. The poUen cutinase showed an optimum pH near 6.5 and was inhibited by thiol-directed reagents such as phydroxymercurnbenzoate and N-ethyl maleimide but not by diisopropylfluoropbosphate, an "active serine"-directed reagent indicating that the polen enzyme is an "-SH cutinase" unlike the fungal enzyme which is a serine cutinase. Excretion of the pollen cutinse into the extracelular fluid was complete within 4 to 6 bourn at 30 C. Since actinomycin D and cyclohexinide showed little effect on the level of cutinase excreted, it appears that cutinase is an enzyme synthesized prior to germination. Release of cutinase into the medium did not require germination. Electron microscopy revealed the presence of a continuous cutin layer on mature stigma with extensive folds, which are proposed to play a role similar to that played by the celular papifine found in the stigma of other plants. Chemical analysis of stigma cutin by depolymerization and combined gas-liquid chromatogrphy and mass spectrometry showed that this cutin consists of mainly the C,6 family of acids. The major (70%) components were dihydroxy C,6 acids which consisted of 10,16-(64%), 9,16-(16%), 8,16-(12%), and 7,16-(8%) dihydroxy palmitic acid. Deuterium-labeling studies sbowed the presence of 16-oxo-9-hydroxy C,6 acid and 16-oxo-10-hydroxy C,6 add in this cutin. The biochemical and ultratctrl studies indicate that the poUen tube may gain entry into stigma using cutinase excreted by the pollen.tal evidence for the presence of cutinase in germinating pollen consisted of the demonstration that addition of cutin preparation to germinating pollen caused a slight increase in titratable acidity (7,18). With this increase as a measure of cutinase, it was found that the presence of cutinase in germinating pollen was a characteristic of plants which have ' cutinized" stigma. Since fungal extracts also catalyzed release of titratable acids in the presence of cutin, including a cutin preparation from stigma surface, it was suggested that cutinase of germinating pollen was "identical with" cutinase detected in molds (7). Cutinase from neither molds nor pollen had been isolated or characterized and, therefore, it was not possible to compare the enzymes from the two sources. Recently, fungal cutinases, isolated in homogeneous form, have been characterized (22, 23. 25). The products released from cutin by the germinating pollen were also not characterized and therefore it was difficult to determine whether the pollen enzyme released all types of monomers of cutin or only the peripheral fatty acids.In this paper we demonstrate, with the use of radioactive cutin, that germinating pollen from nasturtium excrete an enzyme(s) which releases all types of monomers from apple cutin. Results which suggest that the appearance of this cutinase activity...

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Section: Effect Of Enzyme Concentration and Time On Hydrolysis Ofsupporting

confidence: 79%

“…Previous structural studies had demonstrated that 16-oxo-, 9 * or 10-hydroxy C16 acid is a component of cutin particularly of young tissues (13). Since detection of such components is facilitated by deuterium labeling, LiAlD4 was used to depolymerize stigma cutin.…”

Section: Effect Of Enzyme Concentration and Time On Hydrolysis Ofmentioning

confidence: 99%

Production of a Novel Extracellular Cutinase by the Pollen and the Chemical Composition and Ultrastructure of the Stigma Cuticle of Nasturtium (Tropaeolum majus)

1977

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“…1 6-Hydroxy[G-3H]hexadecanoic acid and 10,1 6-dihydroxy[G-'H]hexadecanoic acid were prepared by tritium gas exposure according to Wilzbach's method, followed by rigorous purification as previously described (11). The unlabeled 16-hydroxyhexadecanoic acid was a generous gift from Dr. A. P. Tulloch and the 10, 16-dihydroxyhexadecanoic acid was isolated from V. faba cutin as described previously (15).…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore, synthetic 16-oxo-[G-'H] hexadecanoic acid was readily converted to the dicarboxylic acid by the cell-free preparation. These results demonstrate that epidermis of Vicia faba contains an w-hydroxyacid dehydrogenase and an w-oxoacid dehydrogenase.Long chain dicarboxylic acids are produced by animals (3, 4, 25, 26, 29, 30), microbes (10, 16, 17), and higher plants (5,8,9,11,12,26,27,31). In animals, such acids appear to be intermediates in a catabolic pathway, namely -oxidation, which has no known specific functions, although under certain abnormal conditions, such as ketosis, an increased production of dicarboxylic acids has been observed (23,24).…”

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Biosynthesis of Cutin

Kolattukudy¹,

Croteau²,

Walton³

1975

Plant Physiol.

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Long chain dicarboxylic acids are constituents of the protective biopolymers cutin and suberin of plants. Cell-free extracts from the excised epidermis of Vicia faba leaves catalyzed conversion of 16-hydroxy [G-H]hexadecanoic acid to the corresponding dicarboxylic acid with nicotinamide-adenine dinucleotide phosphate as the preferred cofactor. This enzymatic activity, located largely in the 100,000g supernatant fraction, had a pH optimum near 8. This dehydrogenase showed an apparent Km of 1.25 X 10' M and 3.6 X 10-' M for 16-hydroxyhexadecanoic acid and NADP, respectively. Modification of the substrate, either by esterification of the carboxyl group or by introduction of another hydroxyl group at C-10, resulted in a substantial (two-thirds) decrease in the rate of reaction, and hexadecanol was not a good substrate. The enzyme was inhibited by thiol reagents such as N-ethylmaleimide and p-chloromercuribenzoate. The aldehyde intermediate was trapped by the inclusion of dinitrophenyl hydrazine in the reaction mixture, and the 16-oxo compound was regenerated and identified. Furthermore, synthetic 16-oxo-[G-'H] hexadecanoic acid was readily converted to the dicarboxylic acid by the cell-free preparation. These results demonstrate that epidermis of Vicia faba contains an w-hydroxyacid dehydrogenase and an w-oxoacid dehydrogenase.Long chain dicarboxylic acids are produced by animals (3, 4, 25, 26, 29, 30), microbes (10, 16, 17), and higher plants (5,8,9,11,12,26,27,31). In animals, such acids appear to be intermediates in a catabolic pathway, namely -oxidation, which has no known specific functions, although under certain abnormal conditions, such as ketosis, an increased production of dicarboxylic acids has been observed (23,24). In plants, on the other hand, dicarboxylic acids are constituents of the protective polymers, cutin and suberin. Free, very long chain (C,, and C.0) dicarboxylic acids occur also in the surface lipids of '

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“…Rigorous purification of the methyl ester, followed by hydrolysis and repurification of the acid by TLC gave chemically and radiochemically pure [9,10,11 -3H]n-dotriacontanoic acid (3.75 x 10" cpm/mg). Substrates were dispersed in water containing Tween 20 by sonication (10).…”

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Structure and Biosynthesis of Cuticular Lipids

Kolattukudy¹,

Croteau²,

Brown³

1974

Plant Physiol.

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The structure and composition of the cutin monomers from the flower petals of Vicia faba were determined by hydrogenolysis (LiAlH4) or deuterolysis (LiAID4) followed by thin layer chromatography and combined gas-liquid chromatography and niass spectrometry. The major components were 10, 16-di. hvdroxvhexadecanoic acid (79.8%), 9,16-dihydroxyhexadecanoic acid (4.2 % ), 16-hydroxyhexadecanoic acid (4.2 %), 18-hvdroxvoctadecanoic acid (1.6%), and hexadecanoic acid (2.4%). These results show that flower petal cutin is very similar to leaf cutin of V. faba. Developing petals readily incorporated exogenous [1-_4C]palmitic acid into cutin. Direct conversion of the exogeneous acid into 16-hydroxyhexadecanoic acid, 10, 16-dihydroxy-, and 9,16-dihydroxyhexadecanoic acid was demonstrated by radio gas-liquid chromatography of their chemical degradation products. About 1 % of the exogenous J_14C]palmitic acid was incorporated into C27, C29, and C31 nalkanes, which were identified by combined gas-liquid chromatography and mass spectrometry as the major components of the hydrocarbons of V. faba flowers. The radioactivity distribution among these three alkanes (C27, 15 %; C29, 48%; C31, 38%) was similar to the per cent composition of the alkanes (C27, 12%; C29, 43%; Cai, 44%). [1-_4C]Stearic acid was also incorporated into C27, C29, and CG1 n-alkanes in good yield (3 % ). Trichloroacetate, which has been postulated to be an inhibitor of fattv acid elongation, inhibited the conversion of [1.14C]-stearic acid to alkanes, and the inhibition was greatest for the longer alkanes. Developing flower petals also incorporated exogenous C28, C30, and C31 acids into alkanes in 0.5 % to 5 % yields. [G-3H]n-octacosanoic acid (C28) was incorporated into C27. C28, and CG n-alkanes. [G-3H] elongation-decarboxylation mechanism for the biosynthesis of alkanes.Plant cuticle consists of a cross-esterified hydroxytatty acid polymer, cutin, which is embedded in a mixture of relatively nonpolar, very long chain compounds collectively called wax (12,14). In recent years much progress has been made in the determination of the structure of plant cuticular lipids. However, much of the work has been confined to leaves and fruits from which fairly intact cuticles can be isolated. The nature of the protective polymer on other parts of the plant such as flowers and roots still remains unknown. Even though recent work on the biochemistry of the cuticular lipids has enabled us to propose the most probable pathways involved in their biosynthesis, several key enzymatic steps still remain to be elucidated in cell-free preparations (12). If the flower petal synthesizes the same cuticular lipids as those found on the other parts of the plant, it might offer certain advantages as an experimental material for enzyme level studies. The results presented here constitute the first analysis of a flower cutin and show that the cutin on the V. faba flower petals is very similar, if not identical, to that found on the leaves of the same plant.One of the most common c...

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Biosynthesis of a hydroxy fatty acid polymer, cutin. Identification and biosynthesis of 16-oxo-9- or 10-hydroxypalmitic acid, a novel compound in Vicia faba

Cited by 27 publications

References 15 publications

Production of a Novel Extracellular Cutinase by the Pollen and the Chemical Composition and Ultrastructure of the Stigma Cuticle of Nasturtium (Tropaeolum majus)

Production of a Novel Extracellular Cutinase by the Pollen and the Chemical Composition and Ultrastructure of the Stigma Cuticle of Nasturtium (Tropaeolum majus)

Biosynthesis of Cutin

Structure and Biosynthesis of Cuticular Lipids

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