20 research outputs found
BenzoesÀure-Metabolismus in Zellsuspensionskulturen von Sorbus aucuparia
In cell cultures of Sorbus aucuparia (Rosaceae), the activities of benzoic acid biosynthetic enzymes were co-ordinately stimulated by chitosan treatment, resulting in accumulation of the biphenyl phytoalexin aucuparin. The carbon skeleton of this inducible defence compound is formed by biphenyl synthase (BIS) from benzoyl-CoA and three molecules of malonyl-CoA. The formation of benzoyl-CoA proceeds via benzaldehyde as an intermediate. Benzaldehyde dehydrogenase (BD) converts benzaldehyde to benzoic acid. The preferred substrate for BD was benzaldehyde (Km = 49 ”M). BD activity was dependent on the presence of NAD+ as a cofactor (Km = 67 ”M). The enzyme was inhibited by divalent cations, with Cu2+ and Zn2+ being most inhibitory. Benzoic acid is activated by CoA ligase. Using a homology-based approach, a cDNA encoding CoA ligase was cloned. The enzyme shared 71 and 63.5% identities with Sa4CL2 and Sa4CL3, respectively. The highest catalytic efficiencies were found with p-coumaric and caffeic acids. Using radioisotopic assays, the lack of affinity for benzoic acid was demonstrated. Thus, the enzyme was identified as 4CL isoenzyme (Sa4CL1). Its activity was strictly dependent on the presence of a divalent cation, preferably Mg2+. After elicitor treatment, the transcript levels of all three Sa4CLs increased, however, their maximum mRNA levels were lower than the expression rates observed for the L-phenylalanine ammonia-lyase and BIS1 genes. Sa4CL3 and PAL transcripts also accumulated in response to light treatment, whereas BIS1 expression was not affected by irradiation. A fragment of a fourth cDNA encoding CoA ligase was isolated. A full-length cDNA encoding aldehyde dehydrogenase was cloned. The 1512 bp ORF encoded a 54.8 kDa protein. SaALDH transcripts were up-regulated upon elicitation, strongly suggesting its involvement in benzoic acid metabolism. A full-length cDNA encoding PAL with an ORF of 2160 bp was also cloned from elicitor-treated cell cultures.In Zellkulturen von Sorbus aucuparia (Rosaceae) wurden nach Behandlung mit Chitosan die AktivitĂ€ten von BenzoesĂ€ure-bildenden Enzymen koordiniert stimuliert, was zur Akkumulation des Biphenyl-Phytoalexins Aucuparin fĂŒhrte. Der Kohlenstoffgrundkörper dieses induzierbaren Abwehrstoffs wird von der Biphenylsynthase (BIS) aus Benzoyl-CoA und drei MolekĂŒlen Malonyl-CoA gebildet. Benzoyl-CoA wird ĂŒber die Zwischenstufe des Benzaldehyds gebildet, wobei Benzaldehyd von der Benzaldehyd-Dehydrogenase (BD) in BenzoesĂ€ure umgewandelt wird. Das bevorzugte Substrat der BD ist Benzaldehyd (Km = 49 ”M). Die AktivitĂ€t der BD ist abhĂ€ngig von NAD+ als Co-Faktor (Km = 67 ”M). Durch divalente Kationen wird das Enzym gehemmt, am stĂ€rksten durch Cu2+ und Zn2+. BenzoesĂ€ure wird durch eine CoA-Ligase aktiviert. Mit Hilfe der Homologie-Klonierung wurde eine CoA-Ligase-cDNA isoliert, die eine SequenzidentitĂ€t von 71% und 63,5% zu Sa4CL2 bzw. Sa4CL3 aufwies. Die höchste katalytische AktivitĂ€t bestand mit p-CumarsĂ€ure und KaffeesĂ€ure. In Radioisotop-AnsĂ€tzen wurde keine AffinitĂ€t zu BenzoesĂ€ure gefunden. Demnach handelt es sich um ein 4CL-Isoenzym (Sa4CL1). Dessen AktivitĂ€t hing strikt von divalenten Kationen ab, bevorzugt Mg2+. Nach Elicitor-Behandlung stieg der Transkript-Gehalt fĂŒr alle drei Sa4CLs an, aber ihre maximalen mRNA-Konzentrationen waren geringer als die Expressionsraten des L-Phenylalanin-Ammonium-Lyase (PAL)- und BIS1-Gens. Transkripte fĂŒr Sa4CL3 und PAL akkumulierten auch nach Licht-Behandlung, wĂ€hrend die Expression von BIS1 hiervon unberĂŒhrt blieb. Ein Fragment einer vierten CoA-Ligase-cDNA wurde ebenfalls isoliert, genauso wie eine VolllĂ€nge-cDNA fĂŒr Aldehyd-Dehydrogenase. Der offene Leserahmen von 1512 bp kodierte fĂŒr ein 54,8 kDa-Protein. Die SaALDH-Transkription wurde durch Elicitierung hochreguliert, was fĂŒr eine Beteiligung am BenzoesĂ€ure-Metabolismus spricht. AuĂerdem wurde aus Elicitor-behandelten Zellkulturen eine PAL-VolllĂ€nge-cDNA von 2160 bp kloniert
Characterization of the Mycoremediation of <i>n</i>-Alkanes and Branched-Chain Alkanes by Filamentous Fungi from Oil-Polluted Soil Samples in Kazakhstan
For decades, researchers have focused on containing terrestrial oil pollution. The heterogeneity of soils, with immense microbial diversity, inspires them to transform pollutants and find cost-effective bioremediation methods. In this study, the mycoremediation potentials of five filamentous fungi isolated from polluted soils in Kazakhstan were investigated for their degradability of n-alkanes and branched-chain alkanes as sole carbon and energy sources. Dry weight estimation and gas chromatographyâmass spectrometry (GC-MS) monitored the growth and the changes in the metabolic profile during degradation, respectively. Penicillium javanicum SBUG-M1741 and SBUG-M1742 oxidized medium-chain alkanes almost completely through mono- and di-terminal degradation. Pristane degradation by P. javanicum SBUG-M1741 was >95%, while its degradation with Purpureocillium lilacinum SBUG-M1751 was >90%. P. lilacinum SBUG-M1751 also exhibited the visible degradation potential of tetradecane and phytane, whereby in the transformation of phytane, both the mono- and di-terminal degradation pathways as well as α- and Ă-oxidation steps could be described. Scedosporium boydii SBUG-M1749 used both mono- and di-terminal degradation pathways for n-alkanes, but with poor growth. Degradation of pristane by Fusarium oxysporum SBUG-M1747 followed the di-terminal oxidation mechanism, resulting in one dicarboxylic acid. These findings highlight the role of filamentous fungi in containing oil pollution and suggest possible degradation pathways
Molecular Cloning and Characterization of a Xanthone Prenyltransferase from Hypericum calycinum Cell Cultures
In plants, prenylation of metabolites is widely distributed to generate compounds with efficient defense potential and distinct pharmacological activities profitable to human health. Prenylated compounds are formed by members of the prenyltransferase (PT) superfamily, which catalyze the addition of prenyl moieties to a variety of acceptor molecules. Cell cultures of Hypericum calycinum respond to elicitor treatment with the accumulation of the prenylated xanthone hyperxanthone E. A cDNA encoding a membrane-bound PT (HcPT) was isolated from a subtracted cDNA library and transcript preparations of H. calycinum. An increase in the HcPT transcript level preceded hyperxanthone E accumulation in cell cultures of H. calycinum treated with elicitor. The HcPT cDNA was functionally characterized by expression in baculovirus-infected insect cells. The recombinant enzyme catalyzed biosynthesis of 1,3,6,7-tetrahydroxy-8-prenylxanthone through regiospecific Câ8 prenylation of 1,3,6,7-tetrahydroxyxanthone, indicating its involvement in hyperxanthone E formation. The enzymatic product shared significant structural features with the previously reported cholinesterase inhibitor Îł-mangostin. Thus, our findings may offer a chance for semisynthesis of new active agents to be involved in the treatment of Alzheimerâs disease
Cinnamate:CoA Ligase Initiates the Biosynthesis of a Benzoate-Derived Xanthone Phytoalexin in Hypericum calycinum
Differential Expression of Biphenyl Synthase Gene Family Members in Fire-Blight-Infected Apple âHolsteiner Coxâ 1[W][OA]
Fire blight, caused by the bacterium Erwinia amylovora, is a devastating disease of apple (Malus Ă domestica). The phytoalexins of apple are biphenyls and dibenzofurans, whose carbon skeleton is formed by biphenyl synthase (BIS), a type III polyketide synthase. In the recently published genome sequence of apple âGolden Deliciousâ, nine BIS genes and four BIS gene fragments were detected. The nine genes fall into four subfamilies, referred to as MdBIS1 to MdBIS4. In a phylogenetic tree, the BIS amino acid sequences from apple and Sorbus aucuparia formed an individual cluster within the clade of the functionally diverse type III polyketide synthases. cDNAs encoding MdBIS1 to MdBIS4 were cloned from fire-blight-infected shoots of apple âHolsteiner Cox,â heterologously expressed in Escherichia coli, and functionally analyzed. Benzoyl-coenzyme A and salicoyl-coenzyme A were the preferred starter substrates. In response to inoculation with E. amylovora, the BIS3 gene was expressed in stems of cv Holsteiner Cox, with highest transcript levels in the transition zone between necrotic and healthy tissues. The transition zone was the accumulation site of biphenyl and dibenzofuran phytoalexins. Leaves contained transcripts for BIS2 but failed to form immunodetectable amounts of BIS protein. In cell cultures of apple âCox Orange,â expression of the BIS1 to BIS3 genes was observed after the addition of an autoclaved E. amylovora suspension. Using immunofluorescence localization under a confocal laser-scanning microscope, the BIS3 protein in the transition zone of stems was detected in the parenchyma of the bark. Dot-shaped immunofluorescence was confined to the junctions between neighboring cortical parenchyma cells
Molecular Cloning and Characterization of a Xanthone Prenyltransferase from Hypericum calycinum Cell Cultures
Biphenyl 4-Hydroxylases Involved in Aucuparin Biosynthesis in Rowan and Apple Are Cytochrome P450 736A Proteins
Biphenyl and dibenzofuran levels in transition zones.
<p>Concentrations were determined in the transition zones of fire blight-infected stems of 'Conference' and 'Harrow Sweet' at two time points post-inoculation (dpi). Data are average values ± SD of three independent experiments. Compounds are numbered according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158713#pone.0158713.g004" target="_blank">Fig 4</a>.</p
GC-MS analysis of biphenyls and dibenzofurans.
<p>The methanolic extracts studied were prepared from shoots of 'Harrow Sweet' 28 d after either infection with <i>E</i>. <i>amylovora</i> (a) or mock-inoculation (b). Compounds were separated as trimethylsilyl (TMS) derivatives named 1aâ6a. IS, internal standard (4-phenylphenol), <sup>â </sup>sugar derivatives, *fatty acid derivatives.</p