Characterization of two genes encoding the Mycobacterium tuberculosis ribonucleotide reductase small subunit

Abstract

Two nrdF genes, nrdF1 and nrdF2, encoding the small subunit (R2) of ribonucleotide reductase (RR) from Mycobacterium tuberculosis have 71% identity at the amino acid level and are both highly homologous with Salmonella typhimurium R2F. The calculated molecular masses of R2-1 and R2-2 are 36,588 (322 amino acids [aa]) and 36,957 (324 aa) Da, respectively. Western blot analysis of crude M. tuberculosis extracts indicates that both R2s are expressed in vivo. Recombinant R2-2 is enzymatically active when assayed with pure recombinant M. tuberculosis R1 subunit. Both ATP and dATP are activators for CDP reduction up to 2 and 1 mM, respectively. The gene encoding M. tuberculosis R2-1, nrdF1, is not linked to nrdF2, nor is either gene linked to the gene encoding the large subunit, M. tuberculosis nrdE. The gene encoding MTP64 was found downstream from nrdF1, and the gene encoding alcohol dehydrogenase was found downstream from nrdF2. A nrdA(Ts) strain of E. coli (E101) could be complemented by simultaneous transformation with M. tuberculosis nrdE and nrdF2. An M. tuberculosis nrdF2 variant in which the codon for the catalytically necessary tyrosine was replaced by the phenylalanine codon did not complement E101 when cotransformed with M. tuberculosis nrdE. Similarly, M. tuberculosis nrdF1 and nrdE did not complement E101. Activity of recombinant M. tuberculosis RR was inhibited by incubating the enzyme with a peptide corresponding to the 7 C-terminal amino acid residues of the R2-2 subunit. M. tuberculosis is a species in which a nrdEF system appears to encode the biologically active species of RR and also the only bacterial species identified so far in which class I RR subunits are not arranged on an operon.Mycobacterium tuberculosis is an aerobic, gram-positive bacterium containing a high-GϩC-content genome of approximately 4 ϫ 10 6 bp with a relatively long mean generation time of 24 h and a chromosomal replication time (C period) of 11 h (13). M. tuberculosis can remain in a dormant, nonreplicating state in the human host for years and can be reactivated to cause disease under certain immunological and/or environmental conditions (38). The molecular mechanisms that govern these growth patterns and the regulation of DNA synthesis during these growth phases are now the focus of several lines of investigation. DNA polymerase I (14), topoisomerase I (42), and gyrA and gyrB (21, 33) from M. tuberculosis have been cloned and sequenced. In addition, the origins of replication of M. tuberculosis, M. smegmatis, and M. leprae have been analyzed and shown to contain a typical gram-positive pattern of genes, rnpA-rpmH-dnaA-dnaN-recF-gyrB-gyrA (28). Recently, ribonucleotide reductase (RR), potentially the rate-limiting step in DNA replication, was purified from M. tuberculosis, and the gene encoding the large subunit (R1) was isolated, sequenced, and expressed in Escherichia coli (41).The enzymatic reduction of ribonucleotides by RR regulates the deoxynucleotide precursor pool for DNA synthesis in a wide variety of bacteria and ...

Function and Regulation of Class I Ribonucleotide Reductase-Encoding Genes in Mycobacteria

“…Consequently, these findings argue against specialist roles for NrdZ, NrdF1, and NrdB under conditions of genotoxic and nitrosative stress encountered during the course of infection in mice, and thus they differentiate M. tuberculosis from organisms that utilize a multiplicity of RNRs to modulate the provision of dNTPs for DNA replication and repair under variable and hostile environmental conditions. Instead, our observations have revealed a potential vulnerability in dNTP provision in M. tuberculosis, thereby establishing a compelling rationale for the pursuit of the NrdEF2 form of RNR as a target for antitubercular drug discovery (45,69).…”

Ribonucleotide Reductases

“…Mycobacterium tuberculosis contains the nrdHIE cluster and two copies of the nrdF gene in separate locations on the chromosome (19,108). In Mycoplasma species the organization is nrdFIE (21,22).…”

Staphylococcus aureusNrdH Redoxin Is a Reductant of the Class Ib Ribonucleotide Reductase

“…Many other bacteria such as Escherichia coli (16), Lactobacillus lactis (17), and Mycobacterium and Corynebacterium spp. possess class Ib RNR operons that contain a fourth gene, nrdH (30,44,50), whose product, NrdH, is a thiol-disulfide redoxin (16,17,40,43,49). More-complex situations are found for some bacteria, where the class Ib RNR operon may be duplicated and one or more of the nrdI and nrdH genes may be missing or located in another part of the chromosome (29).…”