Plant sterol metabolism studies on the substrate specificity of an enzyme capable of opening the cyclopropane ring of cycloeucalenol

Heintz, R.; Bimpson, T.; Benveniste, Pierre

doi:10.1016/0006-291x(72)90484-6

Cited by 11 publications

(4 citation statements)

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“…The major uncertainty with using SMY10 as a complementation host for this experiment was that 4,4-dimethyl cyclopropyl sterols such as cycloartenol are not measurably metabolized by higher plant isomerases (8,9,32). For example, the bramble isomerase accepts cycloeucalenol and other 4-desmethyl sterols but has little activity on cycloartenol (9).…”

Section: Cloning Of Cycloeucalenol Cycloisomerase 13395mentioning

confidence: 99%

“…Although the enzyme has not been purified to homogeneity, cycloeucalenol cycloisomerase (also known as cycloeucalenolobtusifoliol isomerase) activity has been observed directly in both dicots (9) and monocots (10). In addition, the presence of an isomerase has been demonstrated indirectly in diverse plants (11)(12)(13)(14) and the protozoan Acanthamoeba polyphaga (15) by identifying cyclopropyl sterols after treatment with isomerase inhibitors.…”

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confidence: 99%

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Functional Cloning of an Arabidopsis thalianacDNA Encoding Cycloeucalenol Cycloisomerase

Lovato¹,

Segura²,

Giner³

et al. 2000

Journal of Biological Chemistry

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Plants and certain protists use cycloeucalenol cycloisomerase (EC 5.5.1.9) to convert pentacyclic cyclopropyl sterols to conventional tetracyclic sterols. We used a novel complementation strategy to clone a cycloeucalenol cycloisomerase cDNA. Expressing an Arabidopsis thaliana cycloartenol synthase cDNA in a yeast lanosterol synthase mutant provided a sterol auxotroph that could be genetically complemented with the isomerase. We transformed this yeast strain with an Arabidopsis yeast expression library and selected sterol prototrophs to obtain a strain that accumulated biosynthetic ergosterol. The novel phenotype was conferred by an Arabidopsis cDNA that potentially encodes a 36-kDa protein.We expressed this cDNA (CPI1) in Escherichia coli and showed by gas chromatography-mass spectrometry that extracts from this strain isomerized cycloeucalenol to obtusifoliol in vitro. The cDNA will be useful for obtaining heterologously expressed protein for catalytic studies and elucidating the in vivo roles of cyclopropyl sterols.Even distantly diverged organisms generally biosynthesize shared molecules by such similar routes that bacteria and yeast have been suitable systems for deducing much of human metabolism. Sterol biosynthesis provides an exception to this trend. Eukaryotes use two distinct pathways ( Fig. 1) to make structurally similar sterols (1). Animals and fungi cyclize oxidosqualene to the tetracyclic ⌬ 8 -triterpene lanosterol (2), which they metabolize further to tetracyclic membrane sterols (3)(4)(5). Plants cyclize instead to the pentacyclic cycloartenol (6, 7) and consequently must isomerize the cyclopropane ring to form ⌬ 8 tetracycles en route to tetracyclic sterols. Why plants use this two-enzyme route (8) rather than cyclizing directly to tetracyclic sterols using lanosterol synthase remains unknown.Although the enzyme has not been purified to homogeneity, cycloeucalenol cycloisomerase (also known as cycloeucalenolobtusifoliol isomerase) activity has been observed directly in both dicots (9) and monocots (10). In addition, the presence of an isomerase has been demonstrated indirectly in diverse plants (11)(12)(13)(14) and the protozoan Acanthamoeba polyphaga (15) by identifying cyclopropyl sterols after treatment with isomerase inhibitors. In D 2 O, the isomerase adds a deuterium to C-19 and abstracts hydrogen from C-8 (16, 17). Neither ATP nor NADPH enhances activity (8), and acidic ring opening seems the most likely mechanism. Because the nonenzymatic acid-catalyzed isomerization (18) requires severe conditions (10% H 2 SO 4 in refluxing isopropyl alcohol for 24 h), it is likely that the enzyme employs a metal or an unusually acidic amino acid residue to open the ring.As a prelude to investigating its catalytic mechanism and biological function, we have cloned and heterologously expressed the Arabidopsis thaliana cDNA that encodes cycloeucalenol cycloisomerase. EXPERIMENTAL PROCEDURESCloning the Cycloeucalenol Cycloisomerase cDNA-The A. thaliana CAS1 1 cDNA (19) cloned into the integrative yeast exp...

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Section: Cloning Of Cycloeucalenol Cycloisomerase 13395mentioning

confidence: 99%

mentioning

confidence: 99%

Functional Cloning of an Arabidopsis thalianacDNA Encoding Cycloeucalenol Cycloisomerase

Lovato¹,

Segura²,

Giner³

et al. 2000

Journal of Biological Chemistry

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“…In plants, an enzyme from bramble tissues was reported (Heintz et al, 1972a) which can open the 9,19-cyclopropane ring of cycloeucalenol, converting it to obtusifoliol. 4,4-dimethyl sterols, cycloartenol sind 24methylene cycloartenol, were found to be very poor substrates for that enzyme (Heintz et al, 1972b). The fungus Fusarium oxysporum, Schlectendahl which is capable of growth utilizing cyclopropanecarboxylic acid as its only source of carbon was able to convert this compound to y-hydroxybutyric acid (Schiller and Chung, 1970a).…”

Section: Resultsmentioning

confidence: 99%

Metabolism of cyclopropane fatty acids by Tetrahymena pyriformis

Al-Shathir

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“…In plants, an enzyme from bramble tissues was reported (Heintz et al, 1972a) which can open the 9,19-cyclopropane ring of cycloeucalenol, converting it to obtusifoliol. 4,4-dimethyl sterols, cycloartenol sind 24-methylene cycloartenol, were found to be very poor substrates for that enzyme (Heintz et al, 1972b). The fungus Fusarium oxysporum, Schlectendahl which is capable of growth utilizing cyclopropanecarboxylic acid as its only source of carbon was able to convert this compound to y-hydroxybutyric acid (Schiller and Chung, 1970a).…”

Section: Resultsmentioning

confidence: 99%

Metabolism of cyclopropane fatty acids by Tetrahymena pyriformis

Al-Shathir

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Plant sterol metabolism studies on the substrate specificity of an enzyme capable of opening the cyclopropane ring of cycloeucalenol

Cited by 11 publications

References 4 publications

Functional Cloning of an Arabidopsis thalianacDNA Encoding Cycloeucalenol Cycloisomerase

Functional Cloning of an Arabidopsis thalianacDNA Encoding Cycloeucalenol Cycloisomerase

Metabolism of cyclopropane fatty acids by Tetrahymena pyriformis

Metabolism of cyclopropane fatty acids by Tetrahymena pyriformis

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