Isolation of ribonucleotide reductase from Mycobacterium tuberculosis and cloning, expression, and purification of the large subunit.
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.
Yang
F
F
Department of Medicine, University of Pennsylvania, Philadelphia.
Lu
G
G
Rubin
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001483:Base Sequence; D003565:Cytidine Diphosphate / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012264:Ribonucleotide Reductases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D000345:Affinity Labels; D011994:Recombinant Proteins; D003565:Cytidine Diphosphate; D000255:Adenosine Triphosphate; D012264:Ribonucleotide Reductases