Abstract
Class I ribonucleotide reductase (RNR) consists of a catalytic subunit (NrdA) and a radical-generating subunit (NrdB) that together catalyse reduction of the four ribonucleotides to their corresponding deoxyribonucleotides. Facklamia ignava NrdB is an unprecedented fusion protein with N-terminal add-ons of a glutaredoxin (Grx) domain followed by an ATP-cone. Grx, which in general is encoded elsewhere in the genome than is the RNR operon, is a known physiological reductant of RNRs. Here we show that the fused Grx domain functions as an efficient reductant of the F. ignava class I RNR via the common dithiol mechanism and interestingly also via a monothiol mechanism, although less efficiently. A Grx that utilizes either or of these two reaction mechanisms has to our knowledge not been observed with a native substrate before. The ATP-cone, which is commonly found as an N-terminal domain of the catalytic subunit of RNRs, is an allosteric on/off switch that promotes dNDP reduction in presence of ATP and inhibits the enzyme activity in presence of dATP. Here we show that dATP bound to the ATP-cone of F. ignava NrdB promotes formation of tetramers that are unable to form enzymatically competent complexes with F. ignava NrdA. The ATP-cone binds two molecules of dATP, but only one molecule of the activating nucleotide ATP. F. ignava NrdB contains the recently identified radical factor Mn2III/IV. We show that NrdA from the firmicute F. ignava can form a catalytically competent RNR with the Mn2III/IV-containing NrdB from the flavobacterium Leeuwenhoekiella blandensis.
Abbreviations and nomenclature
- a-site
- allosteric overall activity site in the ATP-cone
- BSA
- bovine serum albumin
- dNTPs
- deoxyribonucleotides
- DTT
- dithiothreitol
- EPR
- electron paramagnetic resonance
- FPLC
- fast protein liquid chromatography
- GEMMA
- gas-phase electrophoretic macromolecule analysis
- Grx
- glutaredoxin
- GSH
- glutathione
- HED
- 2-hydroxyethyl disulfide
- HIC
- hydrophobic interaction chromatography
- HPLC
- high performance liquid chromatography
- ITC
- isothermal titration calorimetry
- NrdB∆Grx
- NrdB protein lacking 69 N-terminal residues corresponding to the glutaredoxin domain
- NrdB∆169
- NrdB protein lacking the glutaredoxin domain and the ATP-cone domain
- PCR
- polymerase chain reaction
- PMSF
- phenylmethylsulfonyl fluoride
- RNR
- ribonucleotide reductase
- SDS-PAGE
- sodium dodecyl sulphate polyacrylamide gel electrophoresis
- SEC
- size exclusion chromatography
- s-site
- allosteric specificity site in NrdA