An exodeoxyribonuclease from Streptomyces coelicolor: expression, purification and biochemical characterization.
Streptomyces coelicolor A3(2) produces several intra and extracellular enzymes with deoxyribonuclease activities. The examined N-terminal amino acid sequence of one of extracellular DNAases (TVTSVNVNGLL) and database search on S. coelicolor genome showed a significant homology to the putative secreted exodeoxyribonuclease. The corresponding gene (exoSc) was amplified, cloned, expressed in Escherichia coli, purified to homogeneity and characterized. Exonuclease recExoSc degraded chromosomal, linear dsDNA with 3'-overhang ends, linear ssDNA and did not digest linear dsDNA with blunt ends, supercoiled plasmid ds nor ssDNA. The substrate specificity of recExoSc was in the order of dsDNA>ssDNA>3'-dAMP. The purified recExoSc was not a metalloprotein and exhibited neither phosphodiesterase nor RNase activity. It acted as 3'-phosphomonoesterase only at 3'-dAMP as a substrate. The optimal temperature for its activity was 57 degrees C in Tris-HCl buffer at optimal pH=7.5 for either ssDNA or dsDNA substrates. It required a divalent cation (Mg(2+), Co(2+), Ca(2+)) and its activity was strongly inhibited in the presence of Zn(2+), Hg(2+), chelating agents or iodoacetate.
Brnáková
Zuzana
Z
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551 Bratislava, Slovakia. zuzana.brnakova@savba.sk
Godány
Andrej
A
Timko
Jozef
J
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002021:Buffers; D002413:Cations, Divalent / Q000737:chemistry; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D048372:Streptomyces coelicolor / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D002021:Buffers; D002413:Cations, Divalent; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D004279:DNA, Viral; D011994:Recombinant Proteins; D004247:DNA; D000242:Cyclic AMP; D005090:Exodeoxyribonucleases