THE MANGANESE-CONTAINING RIBONUCLEOTIDE REDUCTASE OF CORYNEBACTERIUM AMMONIAGENES IS A CLASS IB ENZYME

Ribonucleotide reductases (RNRs) are key enzymes in living cells that provide the precursors of DNA synthesis, The three characterized class es of RNRs differ by their metal cofactor and their stable organic rad ical. 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 sequenc e 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 clas s 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, demonstra ting that C. ammoniagenes RNR is composed of R1E and R2F components ty pical of class Ib. We also show that the Mn-containing RNR has a speci ficity for the NrdH-redoxin and a response to allosteric effecters tha t are typical of class Ib RNRs. Electron paramagnetic resonance and at omic absorption analyses confirm the presence of Mn as a cofactor and show, for the first time, insignificant amounts of iron and cobalt fou nd in the other classes of RNR. Our discovery that C. ammoniagenes RNR is a class Ib enzyme and possesses all the highly conserved amino aci d side chains that are known to ligate two ferric ions in other class I RNRs evokes new, challenging questions about the control of the meta l site specificity in RNR. The cloning of the entire NrdHIEF locus of C, ammoniagenes will facilitate further studies along these lines.