TY - JOUR T1 - Bio-fermentation of modified flavonoids: an example of in vivo diversification of secondary metabolites JF - Phytochemistry Y1 - 2004 A1 - Michael G Willits A1 - Ma{\"ıté Giovanni A1 - Rogerio T N Prata A1 - Catherine M Kramer A1 - Vincenzo De Luca A1 - John C Steffens A1 - Gerson Graser SP - 31–41 KW - Amino Acid Sequence KW - Arabidopsis KW - Cloning: Molecular KW - Consensus Sequence KW - Escherichia coli KW - Fermentation KW - Flavonoids KW - Glucosyltransferases KW - Mentha piperita KW - Methyltransferases KW - Molecular Sequence Data KW - Onions KW - Phylogeny KW - Recombinant Proteins KW - Sequence Alignment KW - Sequence Homology: Amino Acid KW - Stereoisomerism KW - Substrate Specificity AB - A bio-fermentation technique was used for the in vivo diversification of flavonoid structures based on expression in Escherichia coli of six O-methyltransferases (OMTs) from Mentha x piperita and one O-glucosyltransferase (GT) each from Arabidopsis thaliana and Allium cepa. Enzymes were shown to be regio-specific in in vitro experiments and modified a broad range of flavonoid substrates at various positions. Using the flavonol quercetin as a model substrate, we show that the product spectrum produced with the in vivo approach is identical to that found in vitro. Additionally, using mixed cultures of E. coli expressing different classes of modifying genes (OMTs and GTs), the production of polymethylated flavonoid glucosides was observed. This report demonstrates the potential to increase the structural diversity of plant secondary metabolites using a multi-enzyme, bio-fermentation approach. VL - 65 ER - TY - JOUR T1 - Molecular cloning and biochemical characterization of a novel anthocyanin 5-O-glucosyltransferase by mRNA differential display for plant forms regarding anthocyanin JF - J Biol Chem Y1 - 1999 A1 - M Yamazaki A1 - Z Gong A1 - M Fukuchi-Mizutani A1 - Y Fukui A1 - Y Tanaka A1 - T Kusumi A1 - K Saito SP - 7405–11 KW - Amino Acid Sequence KW - Anthocyanins KW - Cloning: Molecular KW - DNA: Complementary KW - Glucosyltransferases KW - Lamiaceae KW - Molecular Sequence Data KW - Phylogeny KW - Recombinant Proteins KW - RNA: Messenger KW - Saccharomyces cerevisiae KW - Sequence Homology: Amino Acid AB - UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT) is responsible for the modification of anthocyanins to more stable molecules in complexes for co-pigmentation, supposedly resulting in a purple hue. The cDNA encoding 5-GT was isolated by a differential display applied to two different forms of anthocyanin production in Perilla frutescens var. crispa. Differential display was carried out for mRNA from the leaves of reddish-purple and green forms of P. frutescens, resulting in the isolation of five cDNA clones predominantly expressed in the red form. The cDNA encoded a polypeptide of 460 amino acids, exhibiting a low homology with the sequences of several glucosyltransferases including UDP-glucose: anthocyanidin 3-O-glucosyltransferase. By using this cDNA as the probe, we also isolated a homologous cDNA clone from a petal cDNA library of Verbena hybrida. To identify the biochemical function of the encoded proteins, these cDNAs were expressed in Saccharomyces cerevisiae cells. The recombinant proteins in the yeast extracts catalyzed the conversion of anthocyanidin 3-O-glucosides into the corresponding anthocyanidin 3,5-di-O-glucosides using UDP-glucose as a cofactor, indicating the identity of the cDNAs encoding 5-GT. Several biochemical properties (optimum pH, Km values, and sensitivity to inhibitors) were similar to those reported previously for 5-GTs. Southern blot analysis indicated the presence of two copies of 5-GT genes in the genome of both red and green forms of P. frutescens. The mRNA accumulation of the 5-GT gene was detected in the leaves of the red form but not in those of the green form and was induced by illumination of light, as observed for other structural genes for anthocyanin biosynthesis in P. frutescens. VL - 274 ER - TY - JOUR T1 - Characterization of phenylpropene O-methyltransferases from sweet basil: facile change of substrate specificity and convergent evolution within a plant O-methyltransferase family JF - Plant Cell Y1 - 2002 A1 - David R Gang A1 - Noa Lavid A1 - Chloe Zubieta A1 - Feng Chen A1 - Till Beuerle A1 - Efraim Lewinsohn A1 - Joseph P Noel A1 - Eran Pichersky SP - 505–19 KW - Amino Acid Sequence KW - Anisoles KW - DNA: Complementary KW - Escherichia coli KW - Eugenol KW - Evolution: Molecular KW - Gene Expression KW - Gene Expression Regulation: Enzymologic KW - Gene Expression Regulation: Plant KW - Isoflavones KW - Methyltransferases KW - Molecular Sequence Data KW - Molecular Structure KW - Mutagenesis: Site-Directed KW - Mutation KW - Ocimum basilicum KW - Phylogeny KW - Recombinant Proteins KW - Sequence Homology: Amino Acid KW - Substrate Specificity AB - Some basil varieties are able to convert the phenylpropenes chavicol and eugenol to methylchavicol and methyleugenol, respectively. Chavicol O-methyltransferase (CVOMT) and eugenol O-methyltransferase (EOMT) cDNAs were isolated from the sweet basil variety EMX-1 using a biochemical genomics approach. These cDNAs encode proteins that are 90% identical to each other and very similar to several isoflavone O-methyltransferases such as IOMT, which catalyzes the 4'-O-methylation of 2,7,4'-trihydroxyisoflavanone. On the other hand, CVOMT1 and EOMT1 are related only distantly to (iso)eugenol OMT from Clarkia breweri, indicating that the eugenol O-methylating enzymes in basil and C. breweri evolved independently. Transcripts for CVOMT1 and EOMT1 were highly expressed in the peltate glandular trichomes on the surface of the young basil leaves. The CVOMT1 and EOMT1 cDNAs were expressed in Escherichia coli, and active proteins were produced. CVOMT1 catalyzed the O-methylation of chavicol, and EOMT1 also catalyzed the O-methylation of chavicol with equal efficiency to that of CVOMT1, but it was much more efficient in O-methylating eugenol. Molecular modeling, based on the crystal structure of IOMT, suggested that a single amino acid difference was responsible for the difference in substrate discrimination between CVOMT1 and EOMT1. This prediction was confirmed by site-directed mutagenesis, in which the appropriate mutants of CVOMT1 (F260S) and EOMT1 (S261F) were produced that exhibited the opposite substrate preference relative to the respective native enzyme. VL - 14 ER - TY - JOUR T1 - Cloning and expression of UDP-glucose: flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis JF - Planta Y1 - 2000 A1 - M Hirotani A1 - R Kuroda A1 - H Suzuki A1 - T Yoshikawa SP - 1006–13 KW - Amino Acid Sequence KW - Blotting: Northern KW - Chromatography: High Pressure Liquid KW - Cloning: Molecular KW - Escherichia coli KW - Flavonoids KW - Glucosyltransferases KW - Lamiaceae KW - Magnetic Resonance Spectroscopy KW - Molecular Sequence Data KW - Phylogeny KW - Plant Roots KW - Recombinant Proteins AB - A cDNA encoding UDP-glucose: baicalein 7-O-glucosyltransferase (UBGT) was isolated from a cDNA library from hairy root cultures of Scutellaria baicalensis Georgi probed with a partial-length cDNA clone of a UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) from grape (Vitis vinifera L.). The heterologous probe contained a glucosyltransferase consensus amino acid sequence which was also present in the Scutellaria cDNA clones. The complete nucleotide sequence of the 1688-bp cDNA insert was determined and the deduced amino acid sequences are presented. The nucleotide sequence analysis of UBGT revealed an open reading frame encoding a polypeptide of 476 amino acids with a calculated molecular mass of 53,094 Da. The reaction product for baicalein and UDP-glucose catalyzed by recombinant UBGT in Escherichia coli was identified as authentic baicalein 7-O-glucoside using high-performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. The enzyme activities of recombinant UBGT expressed in E. coli were also detected towards flavonoids such as baicalein, wogonin, apigenin, scutellarein, 7,4'-dihydroxyflavone and kaempferol, and phenolic compounds. The accumulation of UBGT mRNA in hairy roots was in response to wounding or salicylic acid treatments. VL - 210 ER -