Abstract
Ribonucleotide reductases (RNRs) are key enzymes in living cells that provide the precursors of DNA synthesis. The three characterized classes of RNRs differ by their metal cofactor and their stable organic radical. We have purified to near homogeneity the enzymatically active Mn-containing RNR of Corynebacterium ammoniagenes, previously claimed to represent a fourth RNR class. N-terminal and internal peptide sequence analyses clearly indicate that the C. ammoniagenes RNR is a class Ib enzyme. In parallel, we have cloned a 10-kilobase pair fragment from C. ammoniagenes genomic DNA, using primers specific for the known class Ib RNR. The cloned class Ib locus contains the nrdHIEF genes typical for class Ib RNR operon. The deduced amino acid sequences of the nrdE and nrdF genes matched the peptides from the active enzyme, demonstrating that C. ammoniagenes RNR is composed of R1E and R2F components typical of class Ib. We also show that the Mn-containing RNR has a specificity for the NrdH-redoxin and a response to allosteric effectors that are typical of class Ib RNRs. Electron paramagnetic resonance and atomic absorption analyses confirm the presence of Mn as a cofactor and show, for the first time, insignificant amounts of iron and cobalt found in the other classes of RNR. Our discovery that C. ammoniagenes RNR is a class Ib enzyme and possesses all the highly conserved amino acid side chains that are known to ligate two ferric ions in other class I RNRs evokes new, challenging questions about the control of the metal site specificity in RNR. The cloning of the entire NrdHIEF locus of C. ammoniagenes will facilitate further studies along these lines.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) Y09572
We report that the Mn-containing Ribonucleotide reductases (RNRs) of C. ammoniagenes is of the class Ib type
PCR Isolation of an Internal Fragment of the C. ammoniagenes nrdF Gene—The deduced amino acid sequences of all known RNR class Ib nrdF genes contain some highly conserved regions that allow the design of NrdF-specific oligonucleotides for PCR amplification
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) Y09572. ¶ Supported by the European Molecular Biology Organisation and the Human Frontier Science Project Organisation. Present address: Institut de Biologie Structurale/CEA-CNRS/Universite Joseph Fourier, 41 Ave. des Martyrs, F-38027 Grenoble Cedex 1, France. The cloning of the entire NrdHIEF locus of C. ammoniagenes will facilitate further studies along these lines. Three well-characterized classes of RNRs, with limited sequence similarities, have been described. They differ in their overall protein structure and cofactor requirement but have in common an allosteric regulation and the use of an organic radical to initiate catalysis through free radical chemistry [1, 2]
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