The Manganese-containing Ribonucleotide Reductase ofCorynebacterium ammoniagenes Is a Class Ib Enzyme

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 Mncontaining 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.Ribonucleotide reductases (RNRs) 1 catalyze the reduction of ribonucleotides providing 2Ј-deoxyribonucleotides for DNA replication and repair. 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) .Apart from the similarity in mechanism, the radical chain initiator and the accompanying metal cofactor differ between the three classes. Class I enzymes (␣ 2 ␤ 2 ) contain a stable tyrosyl radical and a dinuclear iron center. Class II enzymes (␣ or ␣ 2 ) use adenosylcobalamin as cofactor and cleave it to produce a 5Ј-deoxyadenosyl radical (3, 4). The anaerobic class III enzymes (␣ 2 ␤ 2 ) possess a stable glycyl radical and an iron-sulfur cluster (5). Moreover, the different RNRs require their specific physiological reductants thioredoxin, glutaredoxin, and formate, respectively (6 -8). At the beginning of the 1990s, only these three classes of RNR were known, and they were found to cover all major branches of the tree of life. However, additional types of RNRs may remain to be discovered, and questions about non-exhaustively characterized atypical RNRs have to be answered.During the last few years, an additional operon, i...

Ribonucleotide Reductases

“…In the microbial world this is an exception, since the active reductase in other bacteria belongs to subgroup Ib. This also applies to the enzyme from Corynebacterium ammoniagenes that has been reported to depend on manganese (23,53). The only class Ib reductase that has been demonstrated to contain iron is the S. typhimurium enzyme (17), and it is unknown whether the majority of Ib enzymes contain iron or manganese.…”

“…Class Ib exists in many major groups of eubacteria, from the deeply branched Deinococcus genus to enterobacteria. The enzyme from C. ammoniagenes, whose NrdF protein contains manganese, also belongs to this class (23). It is most closely related to the protein from M. tuberculosis (Figure 4).…”

“…In some other bacteria the genes are organized nrdHIEF, as in enterobacteria, with (18,23) or without (20) an nrdH gene. Mycobacterium tuberculosis contains the nrdHIE cluster and two copies of the nrdF gene in separate locations on the chromosome (19,108).…”

“…The importance of this discovery also lies in the subsequent realization that active class I reductases of many other bacteria resemble the new reductase in structure and properties (18)(19)(20)(21)(22)(23). Therefore, two subgroups of class I reductases exist: One (class Ia) has the original E. coli enzyme as the prototype and also includes the eukaryotic enzymes; the second (class Ib) has the new enzyme as its prototype and is present exclusively in bacteria.…”

Crystallization and preliminary X-ray analysis of the small subunit (R2F) of native ribonucleotide reductase fromCorynebacterium ammoniagenes

“…The nature of the Mn-RNR from the Gram-positive bacterium Corynebacterium ammoniagenes ATCC 6872 (Willing et al, 1988;Griepenburg et al, 1998) as a distinct class IV enzyme (Follmann, 2004) has been disputed by its allocation to class Ib from heterologous expression in E. coli (Fieschi et al, 1998;Huque et al, 2000). The C. ammoniagenes Mn-RNR consists of a monomeric catalytic R1E subunit (81 kDa) and a dimeric metallo-cofactor (R2F; 38 kDa) which contains manganese and a tyrosyl radical (Griepenburg et al, 1996).…”