Cloning, sequencing, and expression of the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii.

Abstract

Ribonucleoside-triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacilus kichmannUi, a monomeric adenosylcobalamin-requiring enzyme, catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. The gene for this enzyme has been cloned and sequenced. In contrast to expectations based on mechanistic considerations, there is no statisticafly signicant sequence homology with the Escherichia coli reductase that requires a dinuclear-iron center and tyrosyl radical cofactor. The RTPR has been overexpressed and purified to homogeneity, yielding 90 mg of protein from 2.5 g of bacteria. Initial characterization of the recombinant RTPR indicates that its properties are identical to those of the RTPR isolated from L. kichmannu.Ribonucleotide reductases are uniquely responsible for converting nucleotides to deoxynucleotides in vivo (1-4). In contrast to most enzymes that play essential roles in metabolism in both prokaryotes and eukaryotes, the reductases do not appear, at least superficially, to have been evolutionarily conserved. In Escherichia coli, mammals, and herpesviruses, the enzymes possess two subunits, Rl and R2, each of which is homodimeric (a2132). The R2 subunit possesses a dinuclear iron center-tyrosyl radical cofactor that is essential for nucleotide reduction. The Rl subunit contains the binding sites for the NDP substrates and dNTP allosteric effectors, and the cysteines that are oxidized concomitant with substrate reduction. The ribonucleoside-triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillus leichmannii, the structurally simplest of all known reductases, is a monomer and requires adenosylcobalamin (AdoCbl) as a cofactor (5).Despite these diverse cofactors and quarternary structures, evidence suggests that the E. coli and L. leichmannii enzymes exhibit common patterns ofregulation and chemical mechanisms of reduction. The similarities in the allosteric regulatory properties are evident from investigations of the requirement for specific dNTPs to affect the reduction of specific purine and pyrimidine nucleotides (2, 6, 7). The similarities in mechanism of reduction are evident from investigations employing isotopically labeled nucleotide substrates and from investigations of the mechanisms of inhibition by 2'-chloro-2'-deoxynucleotides (3). With respect to the mechanism of reduction, both enzymes are proposed to initiate catalysis by generation of a protein radical that abstracts the hydrogen atom from the 3' carbon of the nucleotide substrate (3). The difference between the two enzymes is most apparent when considering the manner in which the two distinctly different cofactors give rise to the protein radical. For the E. coli reductase, it is proposed that the tyrosyl radical on the R2 subunit generates by long-range electron transfer a protein radical on the Rl subunit, which initiates turnover of the nucleotide (1, 8). The L. leichmanniiThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby...

Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis

“…3). The immediate source of the nrdJ gene was plasmid pSQUIRE, described previously (25). Predicted native ribosome-binding sites (RBSs) were included in the amplicons for S. sanguinis genes.…”

“…We report the creation of S. sanguinis mutants deleted for nrdHEKF, nrdI, or trxR1 (Fig. 1A), and their characterization by growth studies and by heterologous complementation with the class II nrdJ gene from Lactobacillus leichmannii (25). We also report the testing of the nrdHEKF and nrdI mutants, along with an nrdD mutant lacking the class III RNR, in a rabbit model of endocarditis.…”

The Manganese-containing Ribonucleotide Reductase ofCorynebacterium ammoniagenes Is a Class Ib Enzyme

“…However, a preliminary modeled structure of the C. ammoniagenes R2F protein, based on the E. coli R2 structure, highlights only differences between class Ia and class Ib but none that are specific to the C. ammoniagenes RNR and absent from the other NrdF sequences. 3 The characterization of the C. ammoniagenes RNR as a class Ib enzyme evokes new, challenging questions. The cloning of the NrdHIEF locus will facilitate future studies on this RNR, whereby new insights in the design and fine-tuning of metalactive sites may be gained.…”

“…Class I enzymes (␣ 2 ␤ 2 ) contain a stable tyrosyl radical and a dinuclear iron center. Class II enzymes (␣ or ␣ 2 ) use adenosylcobalamin as cofactor and cleave it to produce a 5Ј-deoxyadenosyl radical (3,4). The anaerobic class III enzymes (␣ 2 ␤ 2 ) possess a stable glycyl radical and an iron-sulfur cluster (5).…”

Alternative oxygen‐dependent and oxygen‐independent ribonucleotide reductases in Streptomyces: cross‐regulation and physiological role in response to oxygen limitation

“…They use coenzyme B12 (adenosylcobalamin) to generate a transient 5 ¢ -deoxyadenosyl radical. The best characterized member of this group, the Lactobacillus leichmannii enzyme, is a monomer of ª 82 kDa (Booker and Stubbe, 1993;Booker et al , 1994;Sintchak et al ., 2002). Despite significant differences in their primary sequence, subunit structure and cofactor usage, these enzymes and the class III RNRs that are confined to bacteria that grow in anaerobic conditions share a common three-dimensional core and possess similar reaction mechanisms (Reichard, 1997;Stubbe et al ., 2001).…”