02941nas a2200349   4500008004100000245016900041210006900210260000800279300001400287490000800301520176300309653002402072653001702096653002302113653002302136653002502159653001402184653002802198653001402226653002502240653001902265653002902284653003402313100001602347700001202363700002402375700001302399700001402412700001402426700001302440856013802453       1999                        eng d00aMolecular cloning and biochemical characterization of a novel anthocyanin 5-O-glucosyltransferase by mRNA differential display for plant forms regarding anthocyanin0 aMolecular cloning and biochemical characterization of a novel an  cMar  a7405–110 v2743 aUDP-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.10aAmino Acid Sequence10aAnthocyanins10aCloning: Molecular10aDNA: Complementary10aGlucosyltransferases10aLamiaceae10aMolecular Sequence Data10aPhylogeny10aRecombinant Proteins10aRNA: Messenger10aSaccharomyces cerevisiae10aSequence Homology: Amino Acid1 aYamazaki, M1 aGong, Z1 aFukuchi-Mizutani, M1 aFukui, Y1 aTanaka, Y1 aKusumi, T1 aSaito, K  uhttps://lamiaceae.myspecies.info/content/molecular-cloning-and-biochemical-characterization-novel-anthocyanin-5-o-glucosyltransferase03120nas a2200349   4500008004100000245018100041210006900222260000800291300001400299490000800313520197500321653002102296653002402317653001702341653002802358653002402386653001402410653001702424653002102441653001102462100001402473700001602487700002702503700001302530700001602543700001302559700001402572700002002586700001402606700001502620856013502635       2001                        eng d00aMalonyl-CoA:anthocyanin 5-O-glucoside-6"'-O-malonyltransferase from scarlet sage (Salvia splendens) flowers. Enzyme purification, gene cloning, expression, and characterization0 aMalonylCoAanthocyanin 5Oglucoside6Omalonyltransferase from scarl  cDec  a49013–90 v2763 aThe orange to blue coloration of flowers in nature is, in most cases, provided by anthocyanins, a class of plant flavonoids, many of which are modified by malonyl group(s). However, the identity of the enzyme catalyzing the malonylation reaction remains to be established. Here, we describe for the first time the purification, characterization, and cDNA cloning of an anthocyanin malonyltransferase from scarlet sage (Salvia splendens) flowers. The purified enzyme (termed Ss5MaT1) was a monomeric 50-kDa protein catalyzing the regiospecific transfer of the malonyl group from malonyl-CoA to the 6"'-hydroxyl group of the 5-glucosyl moiety of anthocyanins. Ss5MaT1 showed a k(cat) value of 7.8 s(-1) at 30 degrees C and pH 7.0 for the malonylation of bisdemalonylsalvianin (pelargonidin 3-(6"-O-caffeyl-beta-glucopyranoside)-5-beta-glucopyranoside) and K(m) values of 101 microm and 57 microm for bisdemalonylsalvianin and malonyl-CoA, respectively. p-Coumaric acid, which mimics an aromatic acyl group linked to the 3-glucosidic moiety of an anthocyanin substrate, was a competitive inhibitor with respect to the substrate. This strongly suggests that the presence of an aromatic acyl group at the 3-glucosidic moiety of anthocyanin is important for substrate recognition by the enzyme. On the basis of the partial amino acid sequences of the purified enzyme, we isolated a cDNA encoding Ss5MaT1. Ss5MaT1 consisted of 462 amino acids and shared motifs that are commonly found among members of a versatile plant acyltransferase family, which was recently shown to include numerous homologs of unknown biochemical functions. Northern blot analysis revealed that the transcripts of Ss5MaT1 were detected in petals, sepals, bracts, and red stems, in accordance with the pigment accumulation patterns. Phylogenetic analysis suggests that the aliphatic and aromatic acylations of anthocyanins are generally catalyzed by subfamily members of the plant acyltransferase family.10aAcyltransferases10aAmino Acid Sequence10aAnthocyanins10aMolecular Sequence Data10aMolecular Structure10aPhylogeny10aPigmentation10aPlant Structures10aSalvia1 aSuzuki, H1 aNakayama, T1 aYonekura-Sakakibara, K1 aFukui, Y1 aNakamura, N1 aNakao, M1 aTanaka, Y1 aYamaguchi, M, A1 aKusumi, T1 aNishino, T  uhttps://lamiaceae.myspecies.info/content/malonyl-coaanthocyanin-5-o-glucoside-6-o-malonyltransferase-scarlet-sage-salvia-splendens