Structure of the Nitrogen-Centered Radical Formed during Inactivation of E. coli Ribonucleotide Reductase by 2‘-Azido-2‘-deoxyuridine-5‘-diphosphate:  Trapping of the 3‘-Ketonucleotide

Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides providing the monomeric precursors required for DNA replication and repair. The class I RNRs are composed of two homodimeric subunits: R1 and R2. R1 has the active site where nucleotide reduction occurs, and R2 contains the diiron tyrosyl radical (Y*) cofactor essential for radical initiation on R1. Mechanism-based inhibitors, such as 2'-azido-2'-deoxyuridine-5'-diphosphate (N(3)UDP), have provided much insight into the reduction mechanism. N(3)UDP is a stoichiometric inactivator that, upon interaction with RNR, results in loss of the Y* in R2 and formation of a nitrogen-centered radical (N*) covalently attached to C225 (R-S-N*-X) in the active site of R1. N(2) is lost prior to N* formation, and after its formation, stoichiometric amounts of 2-methylene-3-furanone, pyrophosphate, and uracil are also generated. On the basis of the hyperfine interactions associated with N*, it was proposed that N* is also covalently attached to the nucleotide through either the oxygen of the 3'-OH (R-S-N*-O-R') or the 3'-C (R-S-N*-C-OH). To distinguish between the proposed structures, the inactivation was carried out with 3'-[(17)O]-N(3)UDP and N* was examined by 9 and 140 GHz EPR spectroscopy. Broadening of the N* signal was detected and the spectrum simulated to obtain the [(17)O] hyperfine tensor. DFT calculations were employed to determine which structures are in best agreement with the simulated hyperfine tensor and our previous ESEEM data. The results are most consistent with the R-S-N*-C-OH structure and provide evidence for the trapping of a 3'-ketonucleotide in the reduction process.

Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

“…␤ is a homodimer that houses the diferric-tyrosyl radical (Y • ) required for initiating nucleotide reduction on ␣. Current evidence suggests that there is one Y • and 2 di-iron clusters/␤2 (19,20). The interactions between ␣ and ␤ are weak (K d of 0.2 M) in the absence of nucleotide (21).…”

Biomimetic Modeling of the Nitrogen-centered Radical Postulated to occur during the Inhibition of Ribonucleotide Reductases by 2'-Azido-2'-deoxynucleotides.

“…1,8 The reduction of ribonucleotides to deoxynucleotides catalyzed by RNRs is initiated by the tyrosyl radical on Tyrosine 122 generating the reactive cysteine thiyl radical at cys439 via a long-range electron transfer process. [17][18][19] . The study of the reduction of uridine 5'-diphosphate (UDP)…”

“…The radical from the cys225-cys462 is then transferred to C3' reducing the keto to hydroxyl with the help of glu441. 1,18 The last step of the mechanism is the regenerating of thiyl radical on cys439 by the radical on C3' abstracting the hydrogen from cys439. 18,24 (Figure 3) Deoxynucleotides then leave the active site allowing for other ribonucleotides to enter and to be reduced by RNRs.…”

“…1,18 The last step of the mechanism is the regenerating of thiyl radical on cys439 by the radical on C3' abstracting the hydrogen from cys439. 18,24 (Figure 3) Deoxynucleotides then leave the active site allowing for other ribonucleotides to enter and to be reduced by RNRs. The cys225-cys462 disulfide unit formed during the reduction is reduced to regenerate the free sulfhydryl groups by the two cysteine residues located at the end of the N-terminal of the R1 protein, cys754 and cys759, in order to continue the mechanistic cycle.…”

“…The regeneration is done by a coenzyme, nicotiamide adenine dinucleotide phosphate (NADPH), acting as a reducing agent donating a hydrogen atom to the disulfide unit. 1,18,25 Figure 3. Acceptable mechanism of the conversion of ribonucleotide to deoxyribonucleotide catalyzed by RNR.…”