B12-dependent ribonucleotide reductases from deeply rooted eubacteria are structurally related to the aerobic enzyme from Escherichia coli.
The ribonucleotide reductases from three ancient eubacteria, the hyperthermophilic Thermotoga maritima (TM), the radioresistant Deinococcus radiodurans (DR), and the thermophilic photosynthetic Chloroflexus aurantiacus, were found to be coenzyme-B12 (class II) enzymes, similar to the earlier described reductases from the archaebacteria Thermoplasma acidophila and Pyrococcus furiosus. Reduction of CDP by the purified TM and DR enzymes requires adenosylcobalamin and DTT. dATP is a positive allosteric effector, but stimulation of the TM enzyme only occurs close to the temperature optimum of 80-90 degrees C. The TM and DR genes were cloned by PCR from peptide sequence information. The TM gene was sequenced completely and expressed in Escherichia coli. The deduced amino acid sequences of the two eubacterial enzymes are homologous to those of the archaebacteria. They can also be aligned to the sequence of the large protein of the aerobic E. coli ribonucleotide reductase that belongs to a different class (class I), which is not dependent on B12. Structure determinations of the E. coli reductase complexed with substrate and allosteric effectors earlier demonstrated a 10-stranded beta/alpha-barrel in the active site. From the conservation of substrate- and effector-binding residues we propose that the B12-dependent class II enzymes contain a similar barrel.
Jordan
A
A
Department of Biochemistry I, Medical Nobel Institute, MBB, Karolinska Institute, S-17177 Stockholm, Sweden.
Torrents
E
E
Jeanthon
C
C
Eliasson
R
R
Hellman
U
U
Wernstedt
C
C
Barbé
J
J
Gibert
I
I
Reichard
P
P
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003038:Cobamides / Q000378:metabolism*; D003565:Cytidine Diphosphate / Q000378:metabolism; D003570:Cytidine Triphosphate / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology / Q000235:genetics; D006095:Gram-Positive Cocci / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D003038:Cobamides; D017931:DNA Primers; D003565:Cytidine Diphosphate; D003570:Cytidine Triphosphate; D012264:Ribonucleotide Reductases; C000913:cobamamide