JoAnne Stubbe

Ribonucleotide reductases: radical enzymes with suicidal tendencies

Ribonucleotide reductases isolated from E. coli and from L. leichmannii differ considerably in their primary and quaternary structures, as well as in their cofactor requirements. Despite these differences, studies with the wt enzymes and the normal substrate, and with the wt enzymes and a variety of mechanism-based inhibitors, demonstrate amazing mechanistic similarities between the two reductases. Recent studies with five cysteine mutants of both reductases reveal strikingly similar phenotypes, indicating that, despite the differences in the primary structures, the groups involved in catalysis in both enzymes appear to be similar.

Mechanism of B12-dependent ribonucleotide reductase

SummaryRibonucleotide reductase from L. leichmmannii catalyzes cleavage of the carbon cobalt bond of AdoCbl homolytically in a kinetically competent fashion. This cleavage triggers a chain of events which results in cleavage of the 3′C-H bond of the nucleotide substrate followed by cleavage of the 2′ carbon hydroxyl bond. Involvement of a radical cation has been suggested as a possible mechanism by which this unusual reduction reaction might occur. Furthermore, cleavage of the 3′ carbon hydrogen bond of [3′-3H]NTP resulted in no 3H release to solvent and no 3H recovered in AdoCbl. These results were interpreted to mean that in this system AdoCbl does not serve as a H abstractor, but rather as a radical chain initiator. A protein residue on the RTPR is postulated to carry out the actual H abstraction from the substrate.These results and the conclusions drawn from them are further supported by recent experiments using [3′-3H]CIUTP. Incubation of RTPR with [3′-3H]CIUTP resulted in release of 3H2O, uracil, PPPi, formation of Coll and 5′ deoxyadenosine. The 3H2O release confirms the enzymes ability to cleave the 3′C-H bond of a nucleotide analog. Furthermore, little if any 3H was recovered in the 5′ deoxyadenosine and the rate of 3H2O release from [3′3H]CIUTP was 12 times faster than the rate of 3H2O release from [5′-3H]AdoCbl. These observations support the conclusions drawn from data with the normal substrate; ie, AdoCbl serves as a radical chain initiator rather than a direct H abstractor from substrate.

Altered Pathway Routing in a Class of Salmonella enterica Serovar Typhimurium Mutants Defective in Aminoimidazole Ribonucleotide Synthetase

In Salmonella enterica serovar Typhimurium, purine nucleotides and thiamine are synthesized by a branched pathway. The last known common intermediate, aminoimidazole ribonucleotide (AIR), is formed from formylglycinamidine ribonucleotide (FGAM) and ATP by AIR synthetase, encoded by the purI gene in S. enterica. Reduced flux through the first five steps of de novo purine synthesis results in a requirement for purines but not necessarily thiamine. To examine the relationship between the purine and thiamine biosynthetic pathways, purI mutants were made (J. L. Zilles and D. M. Downs, Genetics 143:37-44, 1996). Unexpectedly, some mutant purI alleles (R35C/E57G and K31N/A50G/L218R) allowed growth on minimal medium but resulted in thiamine auxotrophy when exogenous purines were supplied. To explain the biochemical basis for this phenotype, the R35C/E57G mutant PurI protein was purified and characterized kinetically. The K(m) of the mutant enzyme for FGAM was unchanged relative to the wild-type enzyme, but the V(max) was decreased 2.5-fold. The K(m) for ATP of the mutant enzyme was 13-fold increased. Genetic analysis determined that reduced flux through the purine pathway prevented PurI activity in the mutant strain, and purR null mutations suppressed this defect. The data are consistent with the hypothesis that an increased FGAM concentration has the ability to compensate for the lower affinity of the mutant PurI protein for ATP.