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 ...

Ribonucleotide reductase, an allosterically regulated, cell cycle-dependent enzyme catalyzing a unique step in the synthesis of DNA, the reduction of 2'-ribonucleotides to 2'-deoxyribonucleotides, was purified 500-fold from Mycobacterium tuberculosis Erdman strain through cell disruption, ammonium sulfate fractionation, and dATP-Sepharose affinity column chromatography. As in eucaryotes and certain bacteria and viruses, the M. tuberculosis enzyme consists of two nonidentical subunits, R1 and R2, both of which are required for activity. R1 has a molecular mass of 84 kDa, as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and photoaffinity labeling with dATP. The amino acid sequences of the N-terminal peptide and two internal peptides were determined, and a partial R1 gene was isolated by PCR with primers designed from these amino acid sequences. Additional coding sequences were isolated by screening size-selected libraries, and a full-length form of M. tuberculosis R1 was generated by PCR amplification of high-molecular-weight M. tuberculosis DNA and expressed in Eschericnia coli. This coding sequence is 2,169 nucleotides long and contains no introns. The predicted molecular mass of R1 from the DNA sequence is 82,244 Da. Recombinant M. tuberculosis R1, purified to homogeneity, was biochemically active when assayed with extracts of M. tuberculosis enriched for R2.

A specific irreversible inhibitor of both cathepsins B and L, Fmoc-Tyr-Ala-CHN2 (FYAD) induced apoptosis of neuroblastoma cells but not other tumor cells. Cysteine protease inhibitors that were not efficient inhibitors of both proteases did not cause death of any cell line tested. Apoptosis was preceded by accumulation of large electron dense vesicles and multivesicular bodies in the cytoplasm. Exposure of cells to the cathepsin D inhibitor, pepstatin, failed to rescue cells from FYAD-induced death. These results indicate that inhibition of cathepsins B and L may provide a unique mechanism for selectively inducing death of neuroblastoma with limited toxicity to normal cells and tissues.

Ribonucleotide reductase (RNR) is a radical enzyme that catalyzes de novo biosynthesis of deoxyribonucleotides. The catalytically required tyrosyl free radical in class I RNRs is produced in conjunction with a µ-oxo-bridged diferric center in protein R2, 35 Å away from the substrate-binding site in protein R1. High-field EPR at 285 GHz was applied to probe the environment of the tyrosyl radical in both native and reconstituted samples of protein R2-2 of class Ib RNR from Mycobacterium tuberculosis. Two distinct peaks (2.0080, 2.0092) of the g x component were observed in the spectrum from freshly purified native R2-2 protein as well as from ferrous iron/oxygen reconstituted apoprotein R2-2. The g x ) 2.0092 peak was relatively stable, whereas the g x ) 2.0080 peak decayed after freezing-thawing and storage. The two peaks corresponding to total g-anisotropies of 0.007 and 0.006, respectively, are interpreted in terms of a non-H-bonded and a weakly H-bonded fraction of the radicals. The hydrogen bond may be provided from a well-ordered water molecule in the vicinity of the radical-iron site, based on a model study of the protein. This first direct observation of conformational heterogeneity of the local tyrosyl radical environment may be important for understanding the role of the radical in the proposed long-range radical transfer through a chain of H-bonded residues from the radical site in protein R2-2 to the catalytic site in protein R1. The enzyme may become activated by connection of a complete H-bonded chain to the radical.