CHARACTERIZATION OF 2 GENES ENCODING THE MYCOBACTERIUM-TUBERCULOSIS RIBONUCLEOTIDE REDUCTASE SMALL-SUBUNIT

Two nrdF genes, nrdF1 and nrdF2, encoding the small subunit (R2) of ri bonucleotide reductase (RR) from Mycobacterium tuberculosis have 71% i dentity at the amino acid level and are both highly homologous with Sa lmonella 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, respect ively, Western blot analysis of crude M. tuberculosis extracts indicat es that both R2s are expressed in vivo. Recombinant R2-2 is enzymatica lly active when assayed with pure recombinant M. tuberculosis R1 subun it. Both ATP and dATP are activators for CDP reduction up to 2 and 1 m M, respectively. The gene encoding M. tuberculosis R2-1, nrdF1, is not linked to nrdF2, nor is either gene linked to the gene encoding the l arge 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) coul d be complemented by simultaneous transformation with M. tuberculosis nrdE and nrdF2. An M. tuberculosis nrdF2 variant in which the codon fo r the catalytically necessary tyrosine was replaced by the phenylalani ne codon did not complement E101 when cotransformed with nl. tuberculo sis nrdE. Similarly, M. tuberculosis nrdF1 and nrdE did not complement E101, Activity of recombinant M. tuberculosis RR was inhibited by inc ubating the enzyme with a peptide corresponding to the 7 C-terminal am ino acid residues of the R2-2 subunit, M. tuberculosis is a species in which a nrdEF system appears to encode the biologically active specie s of RR and also the only bacterial species identified so far in which class I RR subunits are not arranged on an operon.