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Mitogenic factor secreted by Streptococcus pyogenes is a heat-stable nuclease requiring His122 for activity
Makoto Iwasaki1, Hisanaga Igarashi1, Takashi Yutsudo2
1Shionogi Institute for Medical Science, 2-5-1 Mishima, Settsu, Osaka 566, Japan.
Abstract:
The gene encoding a mitogenic factor, termed MF, was cloned from Streptococcus pyogenes and the recombinant MF was overexpressed in Escherichia coli. Both the natural and recombinant MF had heat-resistant nuclease activity. The nuclease activity of MF was characterized using the recombinant protein. MF showed endonuclease activity, digesting ssDNA, dsDNA and tRNA. The optimal pH for the DNase activity of MF was 9.5. The DNase activity was enhanced approximately tenfold by the simultaneous presence of two divalent cations, Mg2+ and Ca2+, compared to either alone and was inhibited by EDTA or NaCl. The heat stability of MF was biphasic; the DNase activity was heat-stable from 0 to 50 degrees C over 80 degrees C but very unstable at around 60 degrees C. DNA digested by MF possessed 5'-phosphorylated and 3'-hydroxylated termini, identical to those obtained by digestion of DNA by pancreatic deoxyribonuclease I. A mutant clone revealed that His122 was a residue essential to the nuclease activity.
Insights
Streptococcus pyogenes mitogenic factor (MF) exhibits heat-stable endonuclease activity, degrading various nucleic acids. Its optimal function requires specific divalent cations and a pH of 9.5.
Area of Science:
- Microbiology
- Molecular Biology
- Enzymology
Background:
- Streptococcus pyogenes produces a mitogenic factor (MF).
- The enzymatic properties of MF, particularly its nuclease activity, were previously uncharacterized.
Purpose of the Study:
- To clone and express the gene encoding MF from Streptococcus pyogenes.
- To characterize the nuclease activity of recombinant MF.
- To identify key residues involved in MF's enzymatic function.
Main Methods:
- Gene cloning and recombinant protein expression in E. coli.
- Biochemical assays to determine nuclease activity, optimal pH, cation requirements, and heat stability.
- Site-directed mutagenesis to investigate essential amino acid residues.
Main Results:
- Recombinant MF demonstrated heat-stable endonuclease activity against single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and transfer RNA (tRNA).
- Optimal activity was observed at pH 9.5, with significant enhancement by Mg2+ and Ca2+ ions.
- MF produced 5'-phosphorylated and 3'-hydroxylated DNA termini, similar to pancreatic DNase I.
- Histidine at position 122 (His122) was identified as crucial for nuclease activity.
Conclusions:
- MF possesses potent endonuclease activity with unique characteristics.
- The enzyme's activity is modulated by pH, divalent cations, and temperature.
- His122 is essential for the catalytic function of MF's nuclease domain.