RESUMO
Pronounced changes in enzymatic patterns occur in petunia tissues when calluses are subcultured on media containing different growth substances. As judged by variations of enzymes related to primary metabolism (6-phosphogluconate and malate dehydrogenases) there are individual responses for each metabolic pathway. Concerning the enzymes of aromatic metabolism: (a) Phenylalanine ammonia-lyase, cinnamate and p-coumarate hydroxylases and the enzyme(s) activating phenylpropanoid units vary in the same manner. (b) Chalcone-flavanone isomerase, a key enzyme in the synthesis of flavonoids, and coniferyl alcohol dehydrogenase, which leads to the monomers of lignins, have, on the other hand, an independent behaviour. These responses show that the enzymes involved in the synthesis and activation of phenylpropanoid units seem to act coordinately in plants. Moreover, the data suggest that the common pathway leading to the activated cinnamic acids and the specific metabolic steps of lignin and flavonoid synthesis are regulated in a different way.
Assuntos
Fenóis/metabolismo , Plantas/enzimologia , Oxirredutases do Álcool/metabolismo , Células Cultivadas , Chalcona , Cinamatos , Cumarínicos , Meios de Cultura , Flavonoides , Liases Intramoleculares , Isomerases/metabolismo , Ligases/metabolismo , Lignina/biossíntese , Malato Desidrogenase/metabolismo , Oxigenases de Função Mista/metabolismo , Fenilalanina Amônia-Liase/metabolismo , Fosfogluconato Desidrogenase/metabolismo , Fatores de TempoRESUMO
The plant aromatic alcohol dehydrogenase, cinnamyl alcohol dehydrogenase (CAD2 from Eucalyptus) was found by sequence analysis of its cloned gene to be homologous to a range of dehydrogenases including alcohol dehydrogenases, L-threonine-3-dehydrogenase, D-xylose reductase and sorbitol dehydrogenase. A homology model of CAD2 was built using the X-ray crystallographic coordinates of horse-liver alcohol dehydrogenase to provide the template, with additional modelling input from other analogous regions of structure from similar enzymes where necessary. The structural model thus produced rationalised the Zn-binding properties of CAD2, indicated the possession of a Rossmann fold (GXGXXG motif), and explained the class A stereospecificity (pro-R hydrogen removal from substrate alcohol) and aromatic substrate specificity of the enzyme. A range of potential ligands was designed based on the homology model and tested as inhibitors of CAD2 and horse liver alcohol dehydrogenase.
Assuntos
Álcool Desidrogenase/química , Oxirredutases do Álcool/química , Lignina/química , Álcool Desidrogenase/antagonistas & inibidores , Oxirredutases do Álcool/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , Eucalyptus/enzimologia , Cavalos , Fígado/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Plantas Medicinais , Estrutura Secundária de Proteína , Alinhamento de SequênciaRESUMO
Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) isoforms were purified from the periderm (containing both suberized and lignified cell layers) of Eucalyptus gunnii Hook stems. Two isoforms (CAD 1P and CAD 2P) were initially characterized, and the major form, CAD 2P, was resolved into three further isoforms by ion-exchange chromatography. Crude extracts contained two aliphatic alcohol dehydrogenases (ADH) and one aromatic ADH, which was later resolved into two further isoforms. Aliphatic ADHs did not use hydroxycinnamyl alcohols as substrates, whereas both aromatic ADH isoforms used coniferyl and sinapyl alcohol as substrates but with a much lower specific activity when compared with benzyl alcohol. The minor form, CAD 1P, was a monomer with a molecular weight of 34,000 that did not co-elute with either aromatic or aliphatic ADH activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis demonstrated that this protein was very similar to another CAD isoform purified from Eucalyptus xylem tissue. CAD 2P had a native molecular weight of approximately 84,000 and was a dimer consisting of two heterogenous subunits (with molecular weights of 42,000 and 44,000). These subunits were differentially combined to give the heterodimer and two homodimers. SDS-PAGE, western blots, and nondenaturing PAGE indicated that the CAD 2P heterodimer was very similar to the main CAD isoform previously purified in our laboratory from differentiating xylem tissue of E. gunnii (D. Goffner, I. Joffroy, J. Grima-Pettenati, C. Halpin, M.E. Knight, W. Schuch, A.M. Boudet [1992] Planta 188: 48-53). Kinetic data indicated that the different CAD 2P isoforms may be implicated in the preferential production of different monolignols used in the synthesis of lignin and/or suberin.
RESUMO
A cell-suspension culture obtained from the hybrid Eucalyptus gunnii/Eucalyptus globulus was hardened by exposure to lower temperatures, whereas in the same conditions cells from a hybrid with a more frost-sensitive genotype, Eucalyptus cypellocarpa/Eucalyptus globulus, were not able to acclimate. During the cold exposure the resistant cells accumulated soluble sugars, in particular fructose and sucrose, with a limited increase in cell osmolality. In contrast, the cell suspension that was unable to acclimate did not accumulate soluble sugars in response to the same cold treatment. To an extent similar to that induced after a cold acclimation, frost-hardiness of the cells increased after a 14-h incubation with specific soluble sugars such as sucrose, raffinose, fructose, and mannitol. Such hardening was also observed for long-term cultures in mannitol-enriched medium. This cryoprotective effect of sugars without exposure to lower temperatures was observed in both the resistant and the sensitive genotypes. Mannitol was one of the most efficient carbohydrates for the cryoprotection of eucalyptus. The best hardiness (a 2.7-fold increase in relative freezing tolerance) was obtained for the resistant cells by the cumulative effect of cold-induced acclimation and mannitol treatment. This positive effect of certain sugars on eucalyptus freezing tolerance was not colligative, since it was independent of osmolality and total sugar content.
RESUMO
Cinnamoyl-coenzyme A:NADP oxidoreductase (CCR, EC 1.2.1.44), the entry-point enzyme into the monolignol biosynthetic pathway, was purified to apparent electrophoretic homogeneity from differentiating xylem of Eucalyptus gunnii Hook. The purified protein is a monomer of 38 kD and has an isoelectric point of 7. Although Eucalyptus gunnii CCR has approximately equal affinities for all possible substrates (p-coumaroyl-coenzyme A, feruloyl-coenzyme A, and sinapoyl-coenzyme A), it is approximately three times more effective at converting feruloyl-coenzyme A than the other substrates. To gain a better understanding of the catalytic regulation of Eucalyptus CCR, a variety of compounds were tested to determine their effect on CCR activity. CCR activity is inhibited by NADP and coenzyme A. Effectors that bind lysine and cysteine residues also inhibit CCR activity. As a prerequisite to the study of the regulation of CCR at the molecular level, polyclonal antibodies were obtained.
RESUMO
Three p-coumarate: CoA ligases were separated from Petunia leaves. There was no interconversion from one form to another. The isoenzymes had a number of common properties: optimum pH, instability in the absence of polyols, action on p-coumaric acid as the common substrate. These enzymes differed significantly with respect to: --their substrate specificity towards the other C6-C3 units of Petunia. Form Ia (caffeate: CoA ligase) acted on caffeic acid, form Ib (sinapate: CoA ligase) on sinapic acid form II (ferulate: CoA ligase) on ferulic acid. --their thermal stability. --their sensitivity to phenolics: (a) caffeate: CoA ligase was inhibited by p-coumaroyl and caffeoyl quinic esters. It was insensitive to p-coumaroyl-glucose, on one hand and to a number of flavonoids on the other. (b) ferulate: CoA ligase was specifically inhibited by naringenin. (c) sinapate: CoA ligase was not inhibited by the selected compounds. In all cases, the inhibition was of the non competitive type and the enzymes were desensized to the modifier action by thermal treatment independently from the enzyme activity. These results suggest the occurrence of distinct sites of reception for the substrate and the inhibitor on the enzyme molecule. All these data are consistent with the hypothesis of the possible participation of each individual form in a limited number of pathways. This would be of physiological interest since the metabolic fate of the different cinnamic acids could be independently controlled at the p-coumarate: CoA ligase level.
Assuntos
Coenzima A Ligases , Fenóis/metabolismo , Plantas/metabolismo , Coenzima A Ligases/metabolismo , Ácidos Cumáricos , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Ligases/isolamento & purificação , Fenóis/farmacologia , Relação Estrutura-AtividadeRESUMO
Oxygen has been shown to be necessary as a cosubstrate for the fungal degradation of lignins. In this work, the active forms of oxygen were tentatively identified in three ways: --effect of chemically generated active radicals and molecular species on lignocellulosic complexes, --use of activated oxygen scavengers in culture media of ligninolytic fungi, --characterization of active forms of oxygen by specific reactions. The data obtained strongly suggest that two main oxygen species are involved, namely OH radical and singlet oxygen (1O2). Chemical or enzymic scavengers inhibit the degradation of lignocelluloses by Phanerochaete chrysosporium. The fungus has been demonstrated to synthesize OH.
Assuntos
Basidiomycota/metabolismo , Celulose/metabolismo , Lignina/metabolismo , Oxigênio/metabolismo , Radicais Livres , Peróxido de Hidrogênio/metabolismoRESUMO
Lignins, which result from the dehydrogenative polymerization of cinnamyl alcohols, are complex heteropolymers deposited in the walls of specific cells of higher plants. Lignins have probably been associated to land colonization by plants but several aspects concerning their biosynthesis, structure and function are still only partially understood. This review focuses on the modern physicochemical methods of structural analysis of lignins, and on the new approaches of molecular biology and genetic engineering applied to lignification. The principles, advantages and limitations of three important analytical tools for studying lignin structure are presented. They include carbon 13 nuclear magnetic resonance, analytical pyrolysis and thioacidolysis. The use of these methods is illustrated by several examples concerning the characterization of grass lignins,'lignin-like'materials in protection barriers of plants and lignins produced by cell suspension cultures. Our present limited knowledge of the spatio temporal deposition of lignins during cell wall differentiation including the nature of the wall components associated to lignin deposition and of the cross-links between the different wall polymers is briefly reviewed. Emphasis is placed on the phenylpropanoid pathway enzymes and their corresponding genes which are described in relation to their potential roles in the quantitative and qualitative control of lignification. Recent findings concerning the promoter sequence elements responsible for the vascular expression of some of these genes are presented. A section is devoted to the enzymes specifically involved in the synthesis of monolignols: cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase. The recent characterization of the corresponding cDNAs/genes offers new possibilities for a better understanding of the regulation of lignification. Finally, at the level of the synthesis, the potential involvement of peroxidases and laccases in the polymerization of monolignols is critically discussed. In addition to previously characterized naturally occurring lignin mutants, induced lignin mutants have been obtained during the last years through genetic engineering. Some examples include plants transformed by O-methyltransferase and cinnamyl alcohol dehydrogenase antisense constructs which exhibit modified lignins. Such strategies offer promising perspectives in gaining a better understanding of lignin metabolism and functions and represent a realistic way to improve plant biomass. Contents Summary 203 I. Introduction 204 II. Main structural features of lignins 205 III. Lignification and cell wall differentiation: spatio-temporal deposition of lignins and inter-relations with other wall components 213 IV. Enzymes and genes involved in the biosynthesis and polymerization of monolignols 216 V. Lignin mutants as a way to improve plant biomass and to explore lignin biochemistry and metabolism 226 VI. Concluding remarks 229 Acknowledgements 230 References 230.
RESUMO
Pathways for hydroxycinnamyl aldehyde incorporation into lignins are revealed by examining transgenic plants deficient in cinnamyl alcohol dehydrogenase, the enzyme that converts hydroxycinnamyl aldehydes to the hydroxycinnamyl alcohol lignin monomers. In such plants the aldehydes incorporate into lignins via radical coupling reactions. As diagnostically revealed by long-range (13)C-(1)H correlative NMR, sinapyl aldehyde (3, 5-dimethoxy-4-hydroxy-cinnamaldehyde) 8-O-4-cross-couples with both guaiacyl (3-methoxy-4-hydroxyphenyl-propanoid) and syringyl (3, 5-dimethoxy-4-hydroxyphenyl-propanoid) units, whereas coniferyl aldehyde cross-couples only with syringyl units.
Assuntos
Cinamatos/metabolismo , Lignina/biossíntese , Lignina/metabolismo , Oxirredutases do Álcool/deficiência , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Aldeídos/química , Aldeídos/metabolismo , Cinamatos/química , Guaiacol/análogos & derivados , Guaiacol/metabolismo , Espectroscopia de Ressonância Magnética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismoRESUMO
Cinnamyl alcohol dehydrogenase (CAD) catalyses the reduction of hydroxycinnamaldehydes (p-coumaryl, coniferyl, sinapyl) to the corresponding alcohols which are the monomeric precursors of lignins. We have demonstrated the occurrence of two isoforms of CAD (CAD1 and CAD2) in bean which differ in terms of subunit Mr, specific activity, substrate affinity and antigenicity. The most abundant polypeptide in bean pods, organs with very limited lignification, is a low affinity CAD isoform (CAD1). This enzyme which is distinct from a benzyl alcohol dehydrogenase with broad substrate specificity, was purified to apparent homogeneity and partial amino acid sequencing was carried out using internal peptides obtained by trypsin cleavage.
Assuntos
Oxirredutases do Álcool/isolamento & purificação , Fabaceae/enzimologia , Isoenzimas/isolamento & purificação , Plantas Medicinais , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Western Blotting , Isoenzimas/metabolismo , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
Two isoforms of laccase were obtained as the predominant phenol-oxidases in defined medium liquid cultures of the "white-rot" fungus Rigidoporus lignosus (R. lignosus). A characterization of the two laccases was made in terms of molecular mass, isoelectric point, metal content and N-terminal sequence. Furthermore, in order to gain information on the structural features related to the metal centers, a study of their geometric arrangement and their redox ability was made. It turned out that the two isoenzymes greatly differed with regard to pH stability, catalytic and copper centers features. It is proposed that all such differences are dependent on the amino acid sequences, which cause a distortion of the copper sites, thus accounting for the redox potential values and kinetic properties.
Assuntos
Basidiomycota/enzimologia , Isoenzimas/química , Isoenzimas/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Sequência de Aminoácidos , Basidiomycota/genética , Domínio Catalítico , Cobre/química , Espectroscopia de Ressonância de Spin Eletrônica , Estabilidade Enzimática , Ponto Isoelétrico , Isoenzimas/genética , Cinética , Lacase , Metais/química , Peso Molecular , Oxirredução , Oxirredutases/genética , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The properties of two enzymes involved in the shikimic acid pathway, dehydroquinate hydro-lyase and shikimate: NADP(+) oxidoreductase were studied in different species of higher plants (pteridophytes, gymnosperms, angiosperms) using chromatography on Sephadex G 100, DEAE cellulose, hydroxylapatite and isoelectric focusing.The two enzymes were not separable by these methods, and we conclude that they exist as an aggregate in higher plants. Moreover, the behaviour of the complex is sometimes quite different according to the species, and these data support the notion of alloenzymes.This situation seems contrary to that found in procaryotic organisms in which all the enzymes of the pathway are separable and in fungi in which five enzymes are associated.These results are discussed in relation to evolution, the channeling function of such complexes and the metabolism of quinic acid in plants.
RESUMO
Quinate:NAD(+) 3-oxidoreductase (EC 1.1.1.24) from carrot cell suspension cultures has previously been shown to be activated by phosphorylation and inactivated by dephosphorylation. Here it is shown that the reactivation of the inactivated quinate:NAD(+) oxidoreductase is an enzyme-mediated process that requires ATP and protein kinase activity. The reactivation is completely inhibited by EGTA and can be restored by the addition of Ca(2+). Cyclic AMP at concentrations up to 5 muM did not have any effect on the reactivation either with or without EGTA in the medium. Calmodulin-depleted fractions containing quinate:NAD(+) oxidoreductase were obtained by passage of the crude extracts through an affinity column of 2-chloro-10-(3-aminopropyl)phenothiazine coupled to Sepharose 4B. The enzyme in this calmodulin-deficient fraction could be inactivated but not reactivated even in the presence of ATP and Ca(2+). However, addition of bovine brain calmodulin completely restored the activity of the enzyme. Half-maximal activation occurred at 130 nM calmodulin. We conclude from these data that the quinate:NAD(+) oxidoreductase is activated by a Ca(2+) - and calmodulin-dependent plant protein kinase.
RESUMO
Three different forms of hydroxycinnamate: CoA ligase (EC 6.2.1-) have been separated by chromatofocusing from poplar stems. These three forms exhibit different substrate specificities and tissue distribution. A correlation was established between the monomeric composition of lignins isolated from xylem and sclerenchyma and the pattern of hydroxycinnamate: CoA ligase isoforms in these tissues. The results obtained indicate that, in poplar, the hydroxycinnamate: CoA ligase isoenzymes could play an important role in the control of the monomeric composition of lignins.
RESUMO
Cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase were purified to apparent homogeneity from poplar stems (Populus euramericana) and their main properties were studied. Only one form was identified for each enzyme. The reductase corresponded to one polypeptide of molecular weight 36 000 and the cinnamyl alcohol dehydrogenase was constituted of two identical subunits of molecular weight 40 000. These characteristics are in agreement with most of the data obtained for the same enzymes isolated from other plants. The two reductive enzymes are inhibited by thiol reagents and a metal chelator 1,10-phenanthroline. The isoelectric point of the reductase (pH 7.5) and of the dehydrogenase (pH 5.6) were determined by chromatofocusing. The cinnamoyl-CoA reductase exhibit a decreasing affinity towards feruloyl-CoA, sinapoyl-CoA and p-coumaroyl-CoA. The cinnamyl alcohol dehydrogenase, which catalyses the reduction of the three cinnamaldehydes, exhibits its highest efficiency towards coniferaldehyde. In spite of differences in the monomeric composition of lignins from xylem and sclerenchyma the reductive enzymes isolated from these two lignified tissues exhibit the same substrate specificity. Consequently, they do not play an important role in the qualitative control of lignins in poplar tissues.
Assuntos
Oxirredutases do Álcool/isolamento & purificação , Aldeído Oxirredutases/isolamento & purificação , Plantas/enzimologia , Oxirredutases do Álcool/antagonistas & inibidores , Aldeído Oxirredutases/antagonistas & inibidores , Quelantes/farmacologia , Fenômenos Químicos , Química , Focalização Isoelétrica , Especificidade por Substrato , Reagentes de Sulfidrila/farmacologia , ÁrvoresRESUMO
Acer pseudoplatanus cell suspension cultures were used to examine the ability of vacuoles isolated from protoplasts to hydrolyze their endogenous proteins. Total cell proteins were labeled by addition of [(3)H]leucine to the culture medium. After preparation of the protoplasts, vacuoles were isolated and were shown to be essentially free from other cellular components. Up to 30% of the [(3)H]leucine-labeled newly synthesized proteins were recovered in the vacuoles. When incubated for 6 hours at 20 degrees C, the vacuoles degraded half of these proteins. The protein breakdown was temperature and pH dependent. Analysis by electrophoresis, in denaturing polyacrylamide gels, revealed that most of the vacuolar proteins were degraded. However, some vacuolar proteins were unaffected during a 6-hour incubation period. The results indicate that vacuoles are able to acquire and degrade intracellular proteins.
RESUMO
Two organic compounds, N-(O-hydroxyphenul)-and N-(O-aminophenyl)sulfinamoyltertiobutyl acetate (OHPAS and NH2PAS, respectively) have been designed for inhibiting cinnamylalcohol dehydrogenase (EC 1.1.1.2), a zinc metalloenzyme involved specifically in lignification. This paper describes their effects in vitro on the activity of the enzyme isolated from poplar and in vivo on the lignification of poplar tissues. The enzyme inhibition was time- and dose-dependent and pseudoirreversible indicating that these compounds could act as suicide inhibitors. Neither OHPAS nor NH2PAS exhibited affinity towards other plant zinc metalloenzymes or phenolic enzymes tested. Only NH2PAS exerted an effect on cinnamoyl: CoA reductase, another specific enzyme of lignification. In addition, these inhibitors, at the concentration of 80 µM, reduced the fluxes of lignin synthesis in poplar stems by 45%. These results show that OHPAS and NH2PAS could become useful tools for reducing lignification in plants.
RESUMO
Guard cell protoplasts from Commelina communis L. illuminated with red light responded to a blue light pulse by an H(+) extrusion which lasted for about 10 minutes. This proton extrusion was accompanied by an O(2) uptake with a 4H(+) to O(2) ratio. The response to blue light was nil in darkness without a preillumination period of red light and increased with the duration of the red light illumination until about 40 minutes. However, acidification in response to a pulse of blue light was obtained in darkness when external NADH (1 millimolar) was added to the incubation medium, suggesting that redox equivalents necessary for the expression of the response to blue light in darkness may be supplied via red light. In accordance with this hypothesis, the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (10 micromolar) decreased the acidification in response to blue light more efficiently when it was added before red light illumination than before the blue light pulse. In the presence of hexacyanoferrate, the acidification in response to a blue light pulse was partly inhibited (53% of control), suggesting a competition for reducing power between ferricyanide reduction and the response to blue light.
RESUMO
Two dehydroquinate hydro-lyases (E.C. 4.2.1.10) have been routinely separated from different organs of Zea mays L. by chromatography on Cellex-D Bio-Rad or hydroxypatite using linear salt gradients. Dehydroquinate hydro-lyase 1 is associated with shikimate: NADP(+) oxidoreductase (E.C. 1.1.1.25). DHQase 2 is a free constitutive enzyme; in this respect it differs from the inducible enzyme of microorganisms which appears only when dehydroquinate or quinate is the principal carbon source. DHQase 1 and DHQase 2 have a similar apparent Michaelis constant and pH optimum, but they differ in their molecular weight, thermal stability and sensitivity to metabolic effectors. DHQase 2 is specifically activated by shikimic acid. This strong activation and the channeling properties of the complex involved in the shikimate pathway can provide an effective means of control in the utilization of dehydroquinate between two different pathways. The significance of such a system involving both a specific regulation of isoenzymes and a molecular compartmentation by means of an enzymatic complex is discussed.
RESUMO
Cell suspension cultures were initiated from two clones of Eucalyptus gunnii differing by their frost resistance.During cold treatments viability of the individual cell lines and of their protoplasts was correlated to the degree of frost resistance of the starting clones.Moreover, at moderate temperature (10°C) the growth rate was higher for the tolerant cells than for the sensitive ones.Free proline content was ten-fold higher in the resistant cell line than in the sensitive one whereas concentrations of other free amino-acids were equivalent.