The anaerobic ribonucleoside triphosphate reductase from Escherichia coli requires S-adenosylmethionine as a cofactor.

Abstract

Extracts from anaerobically grown Escherichia coli contain an oxygen-sensitive activity that reduces CTP to dCTP in the presence of NADPH, dithiothreitol, Mg2e ions, and ATP, different from the aerobic ribonucleoside diphosphate reductase (2'-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) present in aerobically grown E. coli. After fractionation, the activity required at least five components, two heat-labile protein fractions and several low molecular weight fractions. One protein fraction, suggested to represent the actual ribonucleoside triphosphate reductase was purified extensively and on denaturing gel electrophoresis gave rise to several dermed protein bands, all of which were stained by a polyclonal antibody against one of the two subunits (protein Bi) of the aerobic reductase but not by monoclonal anti-B1 antibodies. Peptide mapping and sequence analyses revealed partly common structures between two types of protein bands but also suggested the presence of an additional component. Obviously, the preparations are heterogeneous and the structure of the reductase is not yet established. The second, crude protein fraction is believed to contain several ancillary enzymes required for the reaction. One of the low molecular weight components is S-adenosylmethionine; a second component is a loosely bound metal. We propose that S-adenosylmethionine together with a metal participates in the generation of the radical required for the reduction of carbon 2' of the ribosyl moiety of CTP.Ribonucleotide reductases catalyze the synthesis of the four deoxyribonucleoside triphosphates (dNTPs) required for DNA replication (1-4). The enzymes provide a link between RNA and DNA metabolism. They have attained special biological interest in connection with recent theories concerning the evolution of life on earth since their appearance was a prerequisite for the transition of an "RNA world" into a "DNA world" (5). Several forms of the enzyme occur in nature, with different structures and catalytic mechanisms. Also, their chemistry is of considerable interest. The enzyme from Escherichia coli provided the first example of a biological process that uses a protein radical as a mechanistic option (6, 7). While the direct reduction of ribonucleotides via a radical mechanism is a general mode of dNPT synthesis in all organisms, the way in which this is achieved varies. At present, three classes of ribonucleotide reductases have been described: The aerobic E. coli enzyme (8) is the prototype for class I enzymes (2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1), also present in all higher animal (9) and plant cells (10). These enzymes consist of two proteins: one large homodimer (protein B1 of E. coli) and one small homodimer (protein B2). Characteristic features are the presence of a ferric:tyrosyl radical center in B2 (6) and redox-active dithiols in B1 (11), both involved in the catalytic process. B1 also provides the reductase with allosteric pro...

Two Proteins Mediate Class II Ribonucleotide Reductase Activity in Pseudomonas aeruginosa

“…Class III RNRs, encoded by the nrdD gene, are homodimeric (␣ 2 ) and carry a stable but oxygen-sensitive glycyl radical (9). They require a specific activase, encoded by the nrdG (␤ 2 ) gene, that uses S-adenosylmethionine to generate the glycyl radical (10,11). This class can only operate under anaerobic conditions and has been found in archaea and eubacteria (8).…”

Activation of Class III Ribonucleotide Reductase by Thioredoxin

“…Activation of class III RNR has long been known to be a slow process and to rely upon the obligate presence of DTT in the activation mix (14). However, nothing was known about the function of DTT or the nature of its physiological counterpart.…”

“…Accordingly, in previous studies using DTT and oxidized preparations of protein ␣, activation of the enzyme required prolonged incubation (at least 30 -45 min) (8,9,14). However, as shown in Fig.…”

“…Materials-Enzymes and other components of the anaerobic ribonucleotide reductase system have been obtained as previously described (10,14). Thioredoxin and thioredoxin reductase from E. coli, Chlamydomonas reinhardtii, and Arabidopsis thaliana were provided by Dr. J. Covès (Grenoble, France) and J. P. Jacquot (Nancy, France).…”

“…The reaction was stopped after 20 min by opening the tubes to air and addition of 0.5 ml of 1 M HClO 4 . The solution was then worked up as described earlier (14). One unit of enzyme activity is defined as the formation of 1 nmol of dCTP per min.…”