RESUMEN
Three full-length cDNAs from alfalfa seedlings coding for hydroperoxide lyases were cloned and expressed in Escherichia coli and characterized as cytochrome P450 enzymes. The isoenzymes were specific for 13-hydroperoxy linoleic and linolenic acids and did not use the 9-hydroperoxy isomers as substrates. Because alfalfa contains both specificities, this indicates the presence of two different types of hydroperoxide lyases, each specific for one kind of substrate. The enzymes contain 480 amino acids (54 kDa) and contain an unusual, nonplastidic N-terminal sequence of 22 amino acids, which strongly reduces the enzyme activity. The only known presequence of a hydroperoxide lyase (from Arabidopsis thaliana) was considered to be a transit sequence. The reduced enzyme activity, however, indicates that the hydroperoxide lyases with N-terminal extensions could be pro-enzymes. This hypothesis is supported by the fast release of hydroperoxide lyase products by plants upon wounding. One of the isoenzymes showed a strongly decreased Vmax and Km compared to the other two. Because this is probably due to the substitution of Ser377 by Phe; the residue at position 377 seems to be important. This is the first time that sufficient quantities of hydroperoxide lyase have been obtained for characterization studies, by circumventing difficult purification procedures and degradation of the enzyme. The high expression level, easy purification, good stability and high specificity make these cloned hydroperoxide lyases excellent tools to study the reaction mechanism and structure. We postulate an integrated reaction mechanism, based on the known chemistry of cytochrome P450 enzymes. This is the first mechanism that unifies all observed features of hydroperoxide lyases.
Asunto(s)
Aldehído-Liasas/metabolismo , Sistema Enzimático del Citocromo P-450 , Isoenzimas/metabolismo , Medicago sativa/enzimología , Aldehído-Liasas/química , Aldehído-Liasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Hemo/metabolismo , Isoenzimas/química , Isoenzimas/genética , Cinética , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Análisis EspectralRESUMEN
Sucrose (Suc):Suc 1-fructosyltransferase (1-SST) is the key enzyme in plant fructan biosynthesis, since it catalyzes de novo fructan synthesis from Suc. We have cloned 1-SST from onion (Allium cepa) by screening a cDNA library using acid invertase from tulip (Tulipa gesneriana) as a probe. Expression assays in tobacco (Nicotiana plumbaginifolia) protoplasts showed the formation of 1-kestose from Suc. In addition, an onion acid invertase clone was isolated from the same cDNA library. Protein extracts of tobacco protoplasts transformed with this clone showed extensive Suc-hydrolyzing activity. Conditions that induced fructan accumulation in onion leaves also induced 1-SST mRNA accumulation, whereas the acid invertase mRNA level decreased. Structurally different fructan molecules could be produced from Suc by a combined incubation of protein extract of protoplasts transformed with 1-SST and protein extract of protoplasts transformed with either the onion fructan:fructan 6G-fructosyltransferase or the barley Suc:fructan 6-fructosyltransferase.
Asunto(s)
Fructanos/biosíntesis , Hexosiltransferasas/genética , Cebollas/genética , Secuencia de Aminoácidos , Clonación Molecular , ADN Complementario , Fructanos/química , Glicósido Hidrolasas/genética , Datos de Secuencia Molecular , Cebollas/enzimología , Hojas de la Planta/enzimología , Plantas Tóxicas , Protoplastos/enzimología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Nicotiana/enzimología , beta-FructofuranosidasaRESUMEN
Fructan (polyfructosylsucrose) is an important storage carbohydrate in many plant families. fructan:fructan 6G-fructosyltransferase (6G-FFT) is a key enzyme in the formation of the inulin neoseries, a type of fructan accumulated by members of the Liliales. We have cloned the 6G-FFT from onion by screening a cDNA library using barley sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. The deduced amino acid sequence showed a high homology with plant invertases and 6-SFT. Incubation of protein extracts from transgenic tobacco plants with the trisaccharide 1-kestose and sucrose resulted in the formation of neokestose and fructans of the inulin neoseries with a degree of polymerization up to six. Introduction of the onion 6G-FFT into chicory resulted in the synthesis of fructan of the inulin neoseries, in addition to the synthesis of linear inulin.
Asunto(s)
Allium/enzimología , Fructanos/biosíntesis , Hexosiltransferasas/metabolismo , Inulina/biosíntesis , Plantas Modificadas Genéticamente/metabolismo , Secuencia de Aminoácidos , Cichorium intybus , Biblioteca de Genes , Hexosiltransferasas/biosíntesis , Hexosiltransferasas/química , Hordeum/enzimología , Datos de Secuencia Molecular , Plantas Tóxicas , Protoplastos/enzimología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Nicotiana/enzimología , Vacuolas/enzimologíaRESUMEN
We isolated and characterized two ovule-specific MADS box cDNAs from petunia, designated floral binding protein (fbp) genes 7 and 11. The putative protein products of these genes have approximately 90% of their overall amino acid sequence in common. In situ RNA hybridization experiments revealed that both genes are expressed in the center of the developing gynoecium before ovule primordia are visible. At later developmental stages, hybridization signals were observed only in the ovules, suggesting that these genes are involved in ovule formation. To test this hypothesis, we raised transgenic petunia plants in which both fbp7 and fbp11 expression was inhibited by cosuppression. In the ovary of these transformants, spaghetti-shaped structures developed in positions normally occupied by ovules. These abnormal structures morphologically and functionally resemble style and stigma tissues. Our results show that these MADS box genes belong to a new class of MADS box genes involved in proper ovule development in petunia.