NrdH-redoxin protein mediates high enzyme activity in manganese-reconstituted ribonucleotide reductase from Bacillus anthracis

Abstract

Bacillus anthracis is a severe mammalian pathogen encoding a class Ib ribonucleotide reductase (RNR). RNR is a universal enzyme that provides the four essential deoxyribonucleotides needed for DNA replication and repair. Almost all Bacillus spp. encode both class Ib and class III RNR operons, but the B. anthracis class III operon was reported to encode a pseudogene, and conceivably class Ib RNR is necessary for spore germination and proliferation of B. anthracis upon infection. The class Ib RNR operon in B. anthracis encodes genes for the catalytic NrdE protein, the tyrosyl radical metalloprotein NrdF, and the flavodoxin protein NrdI. The tyrosyl radical in NrdF is stabilized by an adjacent Mn(2)(III) site (Mn-NrdF) formed by the action of the NrdI protein or by a Fe(2)(III) site (Fe-NrdF) formed spontaneously from Fe(2+) and O(2). In this study, we show that the properties of B. anthracis Mn-NrdF and Fe-NrdF are in general similar for interaction with NrdE and NrdI. Intriguingly, the enzyme activity of Mn-NrdF was approximately an order of magnitude higher than that of Fe-NrdF in the presence of the class Ib-specific physiological reductant NrdH, strongly suggesting that the Mn-NrdF form is important in the life cycle of B. anthracis. Whether the Fe-NrdF form only exists in vitro or whether the NrdF protein in B. anthracis is a true cambialistic enzyme that can work with either manganese or iron remains to be established.

FIGURE 1.

UV-visible absorption of B. anthracis Mn-NrdF and Fe-NrdF. The upper panel shows activated Mn-NrdF in the presence of NrdI_ox (trace A), only NrdI_ox (trace B), and enzymatically active Mn-NrdF after separation on anion exchange chromatography (trace C). The lower panel shows Fe-NrdF activated in the presence of NrdI_ox (trace A) and in the absence of NrdI (trace B), and (trace C) tyrosyl radical spectrum (after subtraction of spectrum obtained by incubation with radical scavenger hydroxylamine). The spectra in the upper panel have been normalized to 2 μm NrdF dimer, and in the lower panel, they have been normalized to 10 μm. In the lower panel, all spectra correspond to a radical content of 5.9 μm Tyr^•. A.U. denotes absorbance units.

FIGURE 2.

Interactions between B. anthracis NrdF and NrdI analyzed by GEMMA. The NrdI concentration was 0.006 mg/ml in trace A and 0.012 mg/ml in traces C and E. The NrdF concentrations were 0.01 mg/ml in traces B–E. The composition and predicted sizes of the species (in kDa) are indicated.

FIGURE 3.

GEMMA of B. anthracis Mn-NrdF and Fe-NrdF interactions with NrdE ± dATP. The NrdE concentration was 0.02 mg/ml, the NrdF concentration was 0.01 mg/ml, and the dATP concentration was 50 μm. The composition and predicted sizes of the species (in kDa) are indicated.

FIGURE 4.

GEMMA of NrdH interaction with NrdE ± dATP and NrdF. B. cereus NrdH concentration was 0.004 mg/ml in trace A and 0.008 mg/ml in traces B–D. B. anthracis NrdE was 0.04 mg/ml, B. anthracis Mn-NrdF was 0.02 mg/ml, and dATP was 50 μm. The composition and predicted sizes of the species (in kDa) are indicated.