ISOLATION OF RIBONUCLEOTIDE REDUCTASE FROM MYCOBACTERIUM-TUBERCULOSISAND CLONING, EXPRESSION, AND PURIFICATION OF THE LARGE SUBUNIT

Ribonucleotide reductase, an allosterically regulated, cell cycle-depe ndent enzyme catalyzing a unique step in the synthesis of DNA, the red uction of 2'-ribonucleotides to 2'-deoxyribonucleotides, was purified 500-fold from Mycobacterium tuberculosis Erdman strain through cell di sruption, ammonium sulfate fractionation, and dATP-Sepharose affinity column chromatography. As in eucaryotes and certain bacteria and virus es, 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-polyacrylamid e gel electrophoresis and photoaffinity labeling with dATP. The amino acid sequences of the N-terminal peptide and two internal peptides wer e determined, and a partial R1 gene was isolated by PCR with primers d esigned from these amino acid sequences. Additional coding sequences w ere isolated by screening size-selected libraries, and a full-length f orm of M. tuberculosis R1 was generated by PCR amplification of high-m olecular-weight M. tuberculosis DNA and expressed in Escherichia coli. This coding sequence is 2,169 nucleotides long and contains no intron s. The predicted molecular mass of R1 from the DNA sequence is 82,244 Da. Recombinant M. tuberculosis R1, purified to homogeneity, was bioch emically active when assayed with extracts of M. tuberculosis enriched for R2.