Phenylpropanstoffwechsel

Crystal structure data of cation-dependent catechol O-methyltransferases (COMTs) from mammals and related caffeoyl coenzyme A OMTs (CCoAOMTs) from plants have suggested operative molecular mechanisms. These include bivalent cations that facilitate deprotonation of vicinal aromatic dihydroxy systems and illustrate a conserved arrangement of hydroxyl and carboxyl ligands consistent with the requirements of a metal-activated catalytic mechanism. The general concept of metal-dependent deprotonation via a complexed aspartate is only one part of a more pronounced proton relay, as shown by semiempirical and DFT quantum mechanical calculations and experimental validations. A previously undetected catalytic triad, consisting of Lys157-Asn181-Asp228 residues is required for complete methyl transfer in case of a cation-dependent phenylpropanoid and flavonoid OMT, as described in this report. This triad appears essential for efficient methyl transfer to catechol-like hydroxyl group in phenolics. The observation is consistent with a catalytic lysine in the case of mammalian COMTs, but jettisons existing assumptions on the initial abstraction of the meta-hydroxyl proton to the metal stabilizing Asp154 (PFOMT) or comparable Asp-carboxyl groups in type of cation-dependent enzymes in plants. The triad is conserved among all characterized plant CCoAOMT-like enzymes, which are required not only for methylation of soluble phenylpropanoids like coumarins or monolignol monomers, but is also present in the similar microbial and mammalian cation-dependent enzymes which methylate a comparable set of substrates.

A dsRNAi approach silencing a key enzyme of sinapate ester biosynthesis (UDP-glucose:sinapate glucosyltransferase, encoded by the UGT84A9 gene) in oilseed rape (Brassica napus) seeds was performed to reduce the anti-nutritive properties of the seeds by lowering the content of the major seed component sinapine (sinapoylcholine) and various minor sinapate esters. The transgenic seeds have been produced so far to the T6 generation and revealed a steady suppression of sinapate ester accumulation. HPLC analysis of the wild-type and transgenic seeds revealed, as in the previous generations, marked alterations of the sinapate ester pattern of the transformed seeds. Besides strong reduction of the amount of the known sinapate esters, HPLC analysis revealed unexpectedly the appearance of several minor hitherto unknown rapeseed constituents. These compounds were isolated and identified by mass spectrometric and NMR spectroscopic analyses. Structures of 11 components were elucidated to be 4-O-glucosides of syringate, caffeyl alcohol and its 7,8-dihydro derivative as well as of sinapate and sinapine, along with sinapoylated kaempferol glycosides, a hexoside of a cyclic spermidine alkaloid and a sinapine derivative with an ether-bridge to a C6–C3-unit. These results indicate a strong impact of the transgenic approach on the metabolic network of phenylpropanoids in B. napus seeds. Silencing of UGT84A9 gene expression disrupt the metabolic flow through sinapoylglucose and alters the amounts and nature of the phenylpropanoid endproducts.

Acylation is a prevalent chemical modification that to a significant extent accounts for the tremendous diversity of plant metabolites. To catalyze acyl transfer reactions, higher plants have evolved acyltransferases that accept β-acetal esters, typically 1-O-glucose esters, as an alternative to the ubiquitously occurring CoA-thioester-dependent enzymes. Shared homology indicates that the β-acetal ester-dependent acyltransferases are derived from a common hydrolytic ancestor of the Serine CarboxyPeptidase (SCP) type, giving rise to the name Serine CarboxyPeptidase-Like (SCPL) acyltransferases. We have analyzed structure–function relationships, reaction mechanism and sequence evolution of Arabidopsis 1-O-sinapoyl-β-glucose:l-malate sinapoyltransferase (AtSMT) and related enzymes to investigate molecular changes required to impart acyltransferase activity to hydrolytic enzymes. AtSMT has maintained the catalytic triad of the hydrolytic ancestor as well as part of the H-bond network for substrate recognition to bind the acyl acceptor l-malate. A Glu/Asp substitution at the amino acid position preceding the catalytic Ser supports binding of the acyl donor 1-O-sinapoyl-β-glucose and was found highly conserved among SCPL acyltransferases. The AtSMT-catalyzed acyl transfer reaction follows a random sequential bi-bi mechanism that requires both substrates 1-O-sinapoyl-β-glucose and l-malate bound in an enzyme donor–acceptor complex to initiate acyl transfer. Together with the strong fixation of the acyl acceptor l-malate, the acquisition of this reaction mechanism favours transacylation over hydrolysis in AtSMT catalysis. The model structure and enzymatic side activities reveal that the AtSMT-mediated acyl transfer proceeds via a short-lived acyl enzyme complex. With regard to evolution, the SCPL acyltransferase clade most likely represents a recent development. The encoding genes are organized in a tandem-arranged cluster with partly overlapping functions. With other enzymes encoded by the respective gene cluster on Arabidopsis chromosome 2, AtSMT shares the enzymatic side activity to disproportionate 1-O-sinapoyl-β-glucoses to produce 1,2-di-O-sinapoyl-β-glucose. In the absence of the acyl acceptor l-malate, a residual esterase activity became obvious as a remnant of the hydrolytic ancestor. With regard to the evolution of Arabidopsis SCPL acyltransferases, our results suggest early neofunctionalization of the hydrolytic ancestor toward acyltransferase activity and acyl donor specificity for 1-O-sinapoyl-β-glucose followed by subfunctionalization to recognize different acyl acceptors.

Phenylpropanoid polyamine conjugates have been identified in flowers of many plant species. Their presence in Arabidopsis thaliana has only been recently established in flower buds and pollen grains. Annotation and location of a cation-dependent O-methyltransferase AtTSM1 specifically to the tapetum of young flower buds enabled the subsequent identification of several genes with a putative role in phenylpropanoid polyamine conjugate biosynthesis. Based on the analysis of several A. thaliana knockout mutants, a biosynthetic pathway of these conjugates is proposed, which involves two methylation steps catalyzed by different cation-dependent O-methyltransferases, a cytochrome P450 (CYP98A8) catalyzed hydroxylation, and a conjugating acyl transfer performed by a BAHD-like, hydroxycinnamoyl (HC)-transferase. LC/MS based metabolite profiling of the cyp98A8 knockout line identified new feruloyl- and 4-coumaroylspermidine conjugates in the corresponding flowers consistent with a role of this gene in the hydroxylation of these conjugates. A pattern of minor amounts of bis- and tris-acylspermidine conjugates, likely the products of additional HC-transferases were identified in wild type as well as in the mutant lines. Transcript suppression of the genes early in the pathway was observed in knockout or RNAi-lines of the genes encoding late enzymatic steps. The implication of these findings for spermidine conjugate biosynthesis in flower buds of A. thaliana is discussed.

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Metabolite profiling of soluble primary and secondary metabolites, as well as cell wall-bound phenolic compounds from roots of barrel medic (Medicago truncatula) was carried out by GC–MS, HPLC and LC–MS. These analyses revealed a number of metabolic characteristics over 56 days of symbiotic interaction with the arbuscular mycorrhizal (AM) fungus Glomus intraradices, when compared to the controls, i.e. nonmycorrhizal roots supplied with low and high amounts of phosphate. During the most active stages of overall root mycorrhization, elevated levels of certain amino acids (Glu, Asp, Asn) were observed accompanied by increases in amounts of some fatty acids (palmitic and oleic acids), indicating a mycorrhiza-specific activation of plastidial metabolism. In addition, some accumulating fungus-specific fatty acids (palmitvaccenic and vaccenic acids) were assigned that may be used as markers of fungal root colonization. Stimulation of the biosynthesis of some constitutive isoflavonoids (daidzein, ononin and malonylononin) occurred, however, only at late stages of root mycorrhization. Increase of the levels of saponins correlated AM-independently with plant growth. Only in AM roots was the accumulation of apocarotenoids (cyclohexenone and mycorradicin derivatives) observed. The structures of the unknown cyclohexenone derivatives were identified by spectroscopic methods as glucosides of blumenol C and 13-hydroxyblumenol C and their corresponding malonyl conjugates. During mycorrhization, the levels of typical cell wall-bound phenolics (e.g. 4-hydroxybenzaldehyde, vanillin, ferulic acid) did not change; however, high amounts of cell wall-bound tyrosol were exclusively detected in AM roots.Principal component analyses of nonpolar primary and secondary metabolites clearly separated AM roots from those of the controls, which was confirmed by an hierarchical cluster analysis. Circular networks of primary nonpolar metabolites showed stronger and more frequent correlations between metabolites in the mycorrhizal roots. The same trend, but to a lesser extent, was observed in nonmycorrhizal roots supplied with high amounts of phosphate. These results indicate a tighter control of primary metabolism in AM roots compared to control plants. Network correlation analyses revealed distinct clusters of amino acids and sugars/aliphatic acids with strong metabolic correlations among one another in all plants analyzed; however, mycorrhizal symbiosis reduced the cluster separation and enlarged the sugar cluster size. The amino acid clusters represent groups of metabolites with strong correlations among one another (cliques) that are differently composed in mycorrhizal and nonmycorrhizal roots. In conclusion, the present work shows for the first time that there are clear differences in development- and symbiosis-dependent primary and secondary metabolism of M. truncatula roots.

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Two full-length cDNAs encoding flavonoid-specific glucosyltransferases, UGT73A4 and UGT71F1, were isolated from a cDNA library of Beta vulgaris (Amaranthaceae) cell suspension cultures. They displayed high identity to position-specific betanidin and flavonoid glucosyltransferases from Dorotheanthus bellidiformis (Aizoaceae) and to enzymes with similar substrate specificities from various plant families. The open reading frame of the sequences encode proteins of 476 (UGT73A4) and 492 (UGT71F1) amino acids with calculated molecular masses of 54.07 kDa and 54.39 kDa, and isoelectric points of 5.8 and 5.6, respectively. Both enzymes were functionally expressed in Escherichia coli as His- and GST-tagged proteins, respectively. They exhibited a broad substrate specificity, but a distinct regioselectivity, glucosylating a variety of flavonols, flavones, flavanones, and coumarins. UGT73A4 showed a preference for the 4′- and 7-OH position in the flavonoids, whereas UGT71F1 preferentially glucosylated the 3- or the 7-OH position. Glucosylation of betanidin, the aglycone of the major betacyanin, betanin, in B. vulgaris was also observed to a low extent by both enzymes. Several O-glycosylated vitexin derivatives isolated from leaves of young B. vulgaris plants and rutin obtained from B. vulgaris tissue culture are discussed as potential endogenous products of UGT73A4 and UGT71F1. The results are analyzed with regard to evolution and specificity of plant natural product glucosyltransferases.

From yellow petals of Iceland poppy, besides the known flavonoid gossypitrin, seven kaempferol derivatives were isolated. In addition to kaempferol 3-O-β-sophoroside and kaempferol 3-O-β-sophoroside-7-O-β-glucoside, known from other plants, the mono- and dimalonyl conjugates of the latter were identified by MS and NMR spectroscopy. Structure analyses of a set of co-occurring pigments, the nudicaulins, revealed that they have the identical acylated glycoside moieties attached to a pentacyclic indole alkaloid skeleton for which the structure of 19-(4-hydroxyphenyl)-10H-1,10-ethenochromeno[2,3-b]indole-6,8,18-triol was deduced from MS and NMR as well as chemical and chiroptical methods.