Coenzyme B12-dependent ribonucleotide reductase: evidence for the participation of five cysteine residues in ribonucleotide reduction.

Ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the conversion of ribonucleotides to 2'-deoxyribonucleotides and requires adenosylcobalamin (AdoCbl) as a cofactor. Recent cloning, sequencing, and expression of this protein [Booker, S., & Stubbe, J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8352-8356] have now allowed its characterization by site-directed mutagenesis. The present study focuses on the role of five cysteines postulated to be required for catalysis. The choice of which of the ten cysteines of RTPR were to be mutated was based on extensive studies on the Escherichia coli ribonucleoside diphosphate reductase. Despite the differences between these two reductases in primary sequence, quaternary structure, and cofactor requirements, their mechanisms are strikingly similar. The mutagenesis studies reported herein further suggest that the complex role of the five cysteines is also very similar. A variety of single and double mutants of RTPR were prepared (C731S, C736S, C731 and 736S, C119S, C419S, C408S, and C305S), and their interaction with the normal substrate (CTP) was characterized under several sets of conditions. Mutants C731S, C736S, and C731 and 736S all catalyzed the formation of dCTP at rates similar to those of the wild-type (wt) enzyme in the presence of the artificial reductant DTT. In the presence of the in vivo reducing system (thioredoxin, thioredoxin reductase, and NADPH), however, each of these mutants catalyzed the formation of only 0.6-0.8 dCTPs per mole of enzyme. The inability of these mutants to catalyze multiple turnovers with respect to the in vivo reducing system suggests that their function might be to transfer reducing equivalents from thioredoxin into the active site disulfide of the reductase. Mutants C119S and C419S were targeted as being the active site cysteines, the ones which directly reduce the ribonucleotide substrate. As expected, neither of these mutants catalyzed the formation of dCTP. However, they did catalyze a time-dependent formation of cytosine, destruction of the cofactor, and the appearance of a chromophore associated with the protein--all phenotypes previously observed for the corresponding active site cysteines of the E. coli reductase. Mutant C408S was unable to catalyze dNTP production or cytosine release. Moreover, it was ineffective in catalyzing two additional reactions which are unique to this enzyme: the exchange of tritium from the 5' hydrogens of AdoCbl with H2O and the destruction of AdoCbl under anaerobic conditions to give 5'-deoxyadenosine and cob(II)alamin. These results are consistent with the role of this cysteine as the protein radical responsible for initiating catalysis.