Cysteines involved in radical generation and catalysis of class III anaerobic ribonucleotide reductase. A protein engineering study of bacteriophage T4 NrdD.
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.
Andersson
J
J
Department of Molecular Biology, Stockholm University, SE-10691 Stockholm, Sweden.
Westman
M
M
Sahlin
M
M
Sjoberg
B M
BM
D001426:Bacterial Proteins; D017122:Bacteriophage T4 / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005609:Free Radicals / Q000378:metabolism; D005998:Glycine / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D005609:Free Radicals; D014764:Viral Proteins; D012264:Ribonucleotide Reductases; C101141:anaerobic ribonucleotide reductase; C490361:nrdG protein, Enterobacteria phage T4; C090389:nrdG protein, bacteria; D003545:Cysteine; D010100:Oxygen; D005998:Glycine