Abstract
Class III ribonucleotide reductase (RNR) is an anaerobic glycyl radical enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides. We have investigated the importance in the reaction mechanism of nine conserved cysteine residues in class III RNR from bacteriophage T4. By using site-directed mutagenesis, we show that two of the cysteines, Cys-79 and Cys-290, are directly involved in the reaction mechanism. Based on the positioning of these two residues in the active site region of the known three-dimensional structure of the phage T4 enzyme, and their structural equivalence to two cysteine residues in the active site region of the aerobic class I RNR, we suggest that Cys-290 participates in the reaction mechanism by forming a transient thiyl radical and that Cys-79 participates in the actual reduction of the substrate. Our results provide strong experimental evidence for a similar radical-based reaction mechanism in all classes of RNR but also identify important differences between class III RNR and the other classes of RNR as regards the reduction per se. We also identify a cluster of four cysteines (Cys-543, Cys-546, Cys-561, and Cys-564) in the C-terminal part of the class III enzyme, which are essential for formation of the glycyl radical. These cysteines make up a CX(2)C-CX(2)C motif in the vicinity of the stable radical at Gly-580. We propose that the four cysteines are involved in radical transfer between Gly-580 and the cofactor S-adenosylmethionine of the activating NrdG enzyme needed for glycyl radical generation.
Highlights
Class III ribonucleotide reductase (RNR) is an anaerobic glycyl radical enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides
Based on the positioning of these two residues in the active site region of the known three-dimensional structure of the phage T4 enzyme, and their structural equivalence to two cysteine residues in the active site region of the aerobic class I RNR, we suggest that Cys-290 participates in the reaction mechanism by forming a transient thiyl radical and that Cys-79 participates in the actual reduction of the substrate
Site-directed Mutagenesis—To test our hypothesis that cysteine residues are involved in the reaction mechanism of the class III RNRs, we used site-directed mutagenesis to change conserved cysteines in the T4 NrdD protein
Summary
Based on the positioning of these two residues in the active site region of the known three-dimensional structure of the phage T4 enzyme, and their structural equivalence to two cysteine residues in the active site region of the aerobic class I RNR, we suggest that Cys-290 participates in the reaction mechanism by forming a transient thiyl radical and that Cys-79 participates in the actual reduction of the substrate. We propose that the four cysteines are involved in radical transfer between Gly-580 and the cofactor S-adenosylmethionine of the activating NrdG enzyme needed for glycyl radical generation. Cys-79 from the class III enzyme is in a corresponding position to Cys-225 in R1, the residue in class I RNR that together with Cys-462 forms the redox active cysteine pair mentioned above (Fig. 1). We propose that Cys-290 forms a transient thiyl radical corresponding to Cys-439 in the class I RNR, which suggests that the initiation of the reaction for all three RNR classes is similar. We propose that the Cterminal cysteine cluster is involved in the radical transfer between AdoMet and the stable radical position at Gly-580 in the T4 enzyme
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
