Subunit Interaction Dynamics of Class Ia Ribonucleotide Reductases: In Search of a Robust Assay

Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides (NDP) to deoxynucleotides (dNDP), in part controlling the ratios and quantities of dNTPs available for DNA replication and repair. The active form of E. coli class Ia RNR is an asymmetric α 2 β 2 complex in which α 2 contains the active site and β 2 contains the stable diferric-tyrosyl radical cofactor responsible for initiating the reduction chemistry. Each dNDP is accompanied by disulfide bond formation. We now report that β 2 can act catalytically when assaying RNR in either the absence or presence of an external reductant. In the absence of reductant, rapid chemical quench analysis of a reaction of α 2 (10 μM), substrate, and effector with variable amounts of β 2 (0.1, 1 or 10 μM) yields 3 dCDP/α 2 at all ratios of α 2 :β 2 with a rate constant of 8-9 s −1 , associated with a rate limiting conformational change(s). Stopped-flow fluorescence spectroscopy with a fluorophore-labeled β reveals that the rate constants for subunit association (163 ± 7 μM −1 s −1) and dissociation (75 ± 10 s −1) are fast relative to turnover, consistent with catalytic β 2. When assaying in the presence of an external reducing system, the turnover number is dictated by the ratio of α 2 :β 2 , their concentrations, and the concentration and nature of the reducing system; the

Ribonucleotide reductase, a novel target for gonorrhea

“…Aliquots were quenched at 1, 2, 3 and 4 min in 2% HClO 4 . All samples were neutralized by the addition of 0.5 M KOH and processed following a standard protocol (Ravichandran 2020). Percent inhibition was calculated relative to a 1% DMSO negative control.…”

“…The SA for 0.2 to 10 μM was measured with 1 mM 5-[ 3 H]-CDP, 3 mM ATP and 2 mM NADPH by the discontinuous radioactive assay. Assay detection methods followed the standard protocols (Ge 2003, Ravichandran 2020).…”

“…The diferric tyrosyl radical cofactor was self-assembled with Fe 2+ and O 2 to give 0.7 Y•s/β 2 . Ng His6-tagged RNR subunits as previously described (Ravichandran 2020, Greene 2020).…”

“…These compounds also appear to inhibit class Ia RNRs by a new mode of action, which is altering the enzyme's quaternary structure. The structure for the Ec class Ia RNR is shown with the residues in Ec (23,39) that correspond to the mutation sites in Ng (residues 25 and 41) identified by red boxes (top) (Adapted from Chen 2018). Also shown is the sequence alignment of α in this region for Ec and Ng.…”

“…The structure for the Ec class Ia RNR is shown with the residues in Ec (23, 39) that correspond to the mutation sites in Ng (residues 25 and 41) identified by red boxes (top) (Adapted from Chen 2018). Also shown is the sequence alignment of α in this region for Ec and Ng .…”