RESUMO
An abundant 17 kDa protein which was isolated and characterized from 10-day old healthy root tissue of white lupin (Lupinus albus) proved to have a high sequence similarity to pathogenesis-related proteins found in other species. Subsequently, a corresponding clone (LaPR-10) was identified in a cDNA library prepared from the same tissue that exhibited a high amino acid sequence similarity to a number of the PR-10 family proteins. The clone contains an open reading frame encoding a polypeptide of 158 amino acids, with a predicted molecular mass of 16,905 Da and an isoelectric point of 4.66. Southern blot analysis indicates that LaPR-10 is likely a single-copy gene, or a member of a small gene family. The clone was expressed in Escherichia coli, and its protein product was purified to near homogeneity. Both the native and the recombinant proteins were immunorecognized by antibodies raised against pea PR-10 proteins, and exhibited a ribonucleolytic activity against several RNA preparations, including lupin root total RNA. Characterization of its enzymatic properties indicates that the LaPR-10 protein belongs to the class II ribonucleases. We present evidence that the white lupin 17 kDa protein is constitutively expressed during all stages of root development and, to a lesser extent, in other plant parts. In addition, we demonstrate the presence, in the LaPR-10 amino acid sequence, of a number of motifs that are common to most PR-10 proteins, as well as a RGD motif that is shared only with the alfalfa SRG1 sequence.
Assuntos
Fabaceae/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Plantas Medicinais , Ribonucleases/metabolismo , Sequência de Aminoácidos , Northern Blotting , Southern Blotting , DNA Complementar/química , DNA Complementar/genética , DNA de Plantas/genética , Escherichia coli/genética , Fabaceae/enzimologia , Fabaceae/crescimento & desenvolvimento , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Raízes de Plantas/enzimologia , Raízes de Plantas/crescimento & desenvolvimento , RNA de Plantas/genética , RNA de Plantas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência de AminoácidosRESUMO
We report that the cDNA clone (Accession No. U70424), previously isolated from Arabidopsis thaliana as encoding a caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT) (1), has now been overexpressed in Escherichia coli BL21 and its recombinant protein identified as a novel flavonol 3'-OMT. It is, therefore, renamed AtOMT1. This cDNA clone has previously been identified on the basis of its 88% amino acid sequence similarity and 80% identity to the aspen bispecific lignin OMT (2), the type member of the group involved in lignin biosynthesis. Our data indicate that this novel OMT uses the flavonol quercetin as the preferred substrate, but neither of the hydroxycinnamic acids, caffeic or 5-hydroxyferulic, to any significant extent. This indicates that the high sequence similarity/identity of AtOMT1 to that of the aspen lignin OMT (2) is not sufficient to assign the function of this gene product.
Assuntos
Arabidopsis/enzimologia , Expressão Gênica , Metiltransferases/genética , Metiltransferases/metabolismo , Arabidopsis/genética , Ácidos Cafeicos/metabolismo , Cromatografia Líquida de Alta Pressão , Ácidos Cumáricos/metabolismo , DNA Complementar/genética , Escherichia coli/genética , Cinética , Metiltransferases/química , Filogenia , Quercetina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Termodinâmica , Árvores/enzimologiaRESUMO
A flavonol O-methyltransferase cDNA clone (pF3'OMT) from Chrysosplenium americanum was expressed in Escherichia coli Top 10 and the recombinant protein was purified to near homogeneity by affinity chromatography on chelation resin and gel filtration on Superose 12 columns. The purified protein was enzymatically active as a 42 kDa monomer and exhibited strict specificity for position 3' of 3,7,4'-trimethylquercetin. In did not accept the mono- or dimethyl analogs, the parent aglycone quercetin or the phenylpropanoids, caffeic and 5-hydroxyferulic acids as substrates; thus indicating its involvement in the later steps of polymethylated flavonol synthesis in this plant. The K(m) values of the enzyme for 3,7,4'-trimethylquercetin as substrate and S-adenosyl-L-methionine as co-substrate were 7.2 and 20 microM, respectively. The enzyme activity was strongly inhibited by both Ni2+ and rho-chloromercuribenzoate and was restored by the addition of EDTA or beta-mercaptoethanol, respectively. Antibodies raised against the F3'OMT recombinant protein recognized a protein band migrating at the expected molecular mass of the enzyme on SDS-polyacrylamide gels of protein extracts prepared from various sources. This implies a high degree of structural similarity among these enzymes that is also corroborated by their hydropathy profiles.
