An active ribonucleotide reductase from Arabidopsis thaliana cloning, expression and characterization of the large subunit.
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.
Sauge-Merle
S
S
Laboratoire de Chimie et Biochimie des Centres Rëdox Bioologiques, Université Joseph Fourier, Grenoble, France.
Falconet
D
D
Fontecave
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry; D003001:Cloning, Molecular; D003062:Codon; D003565:Cytidine Diphosphate / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008322:Mammals / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D001426:Bacterial Proteins; D003062:Codon; D018076:DNA, Complementary; D003838:Deoxyadenine Nucleotides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D003565:Cytidine Diphosphate; D000255:Adenosine Triphosphate; D012264:Ribonucleotide Reductases; C026600:2'-deoxyadenosine triphosphate