Cysteines Involved in Radical Generation and Catalysis of Class III Anaerobic Ribonucleotide Reductase

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.

A metal-binding site in the catalytic subunit of anaerobic ribonucleotide reductase

“…In this paper we further investigate the role of the equivalent cysteine residues of the C terminus of E. coli protein ␣ with the help of original site-directed mutants, and confirm that they are critical for glycyl radical formation (5). The E. coli system has the advantage over the bacteriophage one in that it allows pure preparations of the ␤ activase and thus more accurate characterization of the radical generation reaction.…”

“…1), at the C-terminal end of protein ␣ (11). Mutation of any of these cysteines (Cys-543, Cys-546, Cys-561, or Cys-564) to serine in RNR from bacteriophage T4 eliminated production of the glycyl radical, as indirectly shown from the absence of cleavage of the ␣ polypeptide at the radical site during exposure to air (5). In the initial 3D structure of the enzyme from bacteriophage T4, these cysteines were shown to belong to a portion of the C-terminal region of the polypeptide, between the end of the last ␤-strand of the ␣Ϫ␤ barrel and the loop containing the glycyl radical residue (residues 543-570) that was disordered in the crystal (4).…”

“…The crystal structure of the oxygen-stable Gly-580 3 Ala mutant class III RNR (nrdD) from bacteriophage T4 has shown that the glycine site is located at the tip of a loop near the C terminus of the polypeptide, Ϸ5 Å away from an essential cysteine residue (Cys-290 in T4) in the substrate-binding site (4,5). The radical is thus proposed to react with this cysteine to generate an intermediate thiyl radical that serves in turn for abstraction of the H3Ј atom of the ribonucleotide substrate (3,6).…”

Activation of Class III Ribonucleotide Reductase by Thioredoxin

“…In class III RNR, alignments of the 20 nrdD sequences presently available have revealed, among the 15-20 Cys generally present, the occurrence of five invariants, one highly conserved and three moderately conserved cysteine residues (18). Site-directed mutagenesis studies done on the enzyme from the bacteriophage T4 have demonstrated that two of these invariants are directly involved in the turnover of the reaction in agreement with the presence of two cysteines in the substrate site observed in the three-dimensional structure of the protein (12,19). The three other invariants are part of a CXXCX 14 CXXC motif (not visible in the three-dimensional structure) located in the C terminus of the polypeptide.…”

“…The three other invariants are part of a CXXCX 14 CXXC motif (not visible in the three-dimensional structure) located in the C terminus of the polypeptide. Each of these cysteines was essential for the formation of Gly ⅐ and was thus proposed to participate to radical transfer reactions during enzyme activation (19).…”