RESUMO
In all living organisms, deoxyribonucleotides, the DNA precursors, are produced by reduction of the corresponding ribonucleotides catalyzed by ribonucleotide reductase. In mammals as in Escherichia coli, the enzyme consists of two proteins. Protein R1 is the proper reductase as it contains, in the substrate binding site, the reducing active cysteine pair. Protein R2 provides a catalytically essential organic radical. Here we report the cloning, expression, purification and characterization of protein R1 from Arabidopsis thaliana. Expression in E. coli was made possible by coexpression of tRNAArg4 which is required for the utilization of AGA and AGG as codons for arginines. Protein R1 shows extensive similarities with protein R1 from mammals: (a) it shows 69% amino-acid sequence identity to human and mouse R1 protein; (b) it is active during CDP reduction by dithiothreitol, in the presence of protein R2 [Sauge-Merle, S., Laulhère, J.-P., Coves, J., Ménage, S., Le Pape, L. & Fontecave, M. (1997) J. Biol. Inorg. Chem. 2, 586-594]; (c) activity is stimulated by thioredoxin and ATP and is inhibited by dATP, showing that as in the mammalian enzyme, the plant ribonucleotide reductase seems to be allosterically regulated by positive (ATP) and negative (dATP) effectors.
Assuntos
Arabidopsis/enzimologia , Proteínas de Plantas/genética , Ribonucleotídeo Redutases/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Arabidopsis/genética , Proteínas de Bactérias/química , Clonagem Molecular , Códon , Cistina Difosfato/metabolismo , DNA Complementar/genética , Nucleotídeos de Desoxiadenina/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Camundongos , Dados de Sequência Molecular , Oxirredução , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleotídeo Redutases/antagonistas & inibidores , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
We have cloned and sequenced a pea Lhcb3 gene, encoding a Photosystem II chlorophyll a/b-binding protein. Sequence analysis indicates that the gene contains two introns and predicts a polypeptide of 265 amino acids. The predicted polypeptide sequence is highly homologous to the polypeptide sequences deduced from Lhcb3 genes previously characterized in tomato and barley.