Electron paramagnetic resonance (EPR) spectroscopy of the stable-free radical in the native metallo-cofactor of the manganese-ribonucleotide reductase (Mn-RNR) of Corynebacterium glutamicum.
Ribonucleotide reductases (RNR; EC 1.17.4.1) provide the 2'-deoxyribonucleotides for DNA replication of proliferating cells by a uniform radical mechanism using diverse metals. The native metallo-cofactor of the Corynebacterium glutamicum RNR contains manganese and is sensitive to EDTA and radical scavengers. Hybrid holoenzymes, capable of ribonucleotide reduction, were composed of the small manganese-containing (R2F) and the large catalytic subunit (R1E) from either of the two corynebacterial RNRs. A synthetic peptide deduced from the C-terminal region of the nrdF gene inhibited the C. glutamicum-RNR non-competitively and cross-reacted with the C. ammoniagenes-RNR. The C. glutamicum-R2F has a saturable organic radical signal at g=2.005 detected by electron paramagnetic resonance (EPR) spectroscopy and shows a distinct absorption at 408 nm indicative of a tyrosyl-like organic radical (Y.). Quantification of the metal content revealed 0.06 mol Fe but 0.8 mol Mn per mol R2F-monomer and would thus assign two manganese atoms bound to the dimeric metallo-cofactor, while a distinct enzymatic activity (32 micromol x mg(-1) x min(-1)) was observed in the biochemical complementation assay. Divergence of the C. glutamicum-RNR studied here from the prototypical Salmonella typhimurium class 1b enzyme and the Chlamydia trachomatis class Ic enzyme is discussed below.
Abbouni
Bouziane
B
Institut fur Mikrobiologie, Leibniz Universitat Hannover, Hannover, Germany.
Oehlmann
Wulf
W
Stolle
Patrick
P
Pierik
Antonio J
AJ
Auling
Georg
G
D000595:Amino Acid Sequence; D002692:Chlamydia trachomatis / Q000201:enzymology; D048230:Corynebacterium glutamicum / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology
D005609:Free Radicals; D008345:Manganese; D012264:Ribonucleotide Reductases