Characterization of C439SR1, a mutant of Escherichia coli ribonucleotide diphosphate reductase: evidence that C439 is a residue essential for nucleotide reduction and C439SR1 is a protein possessing novel thioredoxin-like activity.
Ribonucleotide reductase from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides. Cysteine 439 is proposed to be the protein radical on R1 which initiates the reduction reaction by cleavage of the 3' carbon-hydrogen bond of the nucleotide (Mao et al., 1992a,b). C439 is thus proposed to be essential for catalysis. The C439S mutant of R1 (C439SR1) was prepared. The structure of this mutant was determined to be similar to wt-R1, based on identical CD spectra, isolation via an affinity column specific for the allosteric binding domain, binding of the substrate GDP, and competition with R1 for binding to R2. Preparations of C439SR1 are contaminated with low levels of wt-R1 due to the expression system. The wt-R1 in these preparations can be specifically inactivated by the stoichiometric mechanism-based inhibitor, 2'-azido-2'-deoxyuridine 5'-diphosphate. The activity of the resulting C439SR1 was shown to be less than 0.03% that of the corresponding wt-R1. This is the lower limit of detection with the present assay method. Thus C439 appears to be essential for catalysis. During these studies an unexpected activity of the C439SR1 was uncovered. Its additional cysteines, presumably C754 and C759, appear to function as a thioredoxin with the wt-R1, even though it is incapacitated with respect to nucleotide reduction.
Mao
S S
SS
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Yu
G X
GX
Chalfoun
D
D
Stubbe
J
J
D001483:Base Sequence; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D009841:Oligonucleotides; D010084:Oxidation-Reduction; D012262:Ribonucleoside Diphosphate Reductase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism*
D009711:Nucleotides; D009841:Oligonucleotides; D013879:Thioredoxins; D012262:Ribonucleoside Diphosphate Reductase; D003545:Cysteine