Reactivation of Serratia marcescens Endonuclease NucSma(H89G) by Hydroxilamine

R.G. Khamidullina, I.I. Fazleyeva, O.A. Gimadutdinow*

Kazan Federal University, Kazan, 420008 Russia

E-mail: *Oleg.Gimadutdinov@kpfu.ru

Received December 21, 2016

Full text PDF

Abstract

Serratia marcescens endonuclease is the most nonspecific nuclease known. This nuclease refers to enzymes that can cleave both DNA and RNA. The active site of this enzyme is characterized by the H–N–H motif, but more important functions have been shown for the first amino acid of this motif in Serratia marcescens nuclease. It is known that histidine is essential in catalytic activity of many   nucleases. Histidine functions as a general base that activates a water molecule for a nucleophilic attack at the diester linkages phosphorus atom and causes its rupture. It has been demonstrated that glycine-histidine substitution in Serratia marcescens endonuclease leads to inactivation of the enzyme. At present, the method of chemical recovery of enzymes has become very widespread. Upon addition of certain low molecular compounds, which are similar in chemical properties to the lost amino acid residues, to the medium with the inactive mutant enzyme, the activity of this enzyme has been restored. The recovery effect has been observed due to the fact that the original amino acids are replaced by a low molecular weight as a result of mutations. In this work we have been able to restore the activity of Serratia marcescens mutant endonuclease by adding hydroxylamine, where histidine at position 89 is replaced by glycine.

Keywords: endonuclease, plasmid, hydroxylamine, histidine, imidazole

Figure Captions

Fig. 1. The structure of pHisNucSma plasmid [4].

Fig. 2. The dependence of optical density D600 on time for E. coli recombinant strains: 1 – E. coli TGE900 NucSma(H89G) without hydroxylamine; 2 – E. coli TGE900 NucSma(H89G) with hydroxylamine; 3 – E. coli TGE900 NucSma(wt) with hydroxylamine; 4 – E. coli TGE900 NucSma(wt) without hydroxylamine.

Fig. 3. The electropherogram of proteins of E. coli TGE900 recombinant strains before and after the induction of S. marcescens endonuclease gene: М –RageRuler Unstained Protein Ladder molecular marker produced by Fermentas; 1 – NucSma(H89G) before the induction of S. marcescens endonuclease gene; 2 – NucSma(H89G) after the induction of S. marcescens endonuclease gene; 3 – NucSma(wt) before the induction of S. marcescens endonuclease gene; 4 – NucSma(wt) after the induction of S. marcescens endonuclease gene.

Fig. 4. The electropherogram of protein fractions after the elution with Ni–NTA agarose: М – RageRuler Unstained Protein Ladder molecular marker produced by Fermentas; 1–3 – NucSma(H89G); 4–6 – NucSma(wt).

Fig. 5. The electropherogram of products of the hydrolysis of pBSK plasmid DNA by S. marcescens endonuclease of the initial and mutant variants in the presence of hydroxylamine: 1 – marker produced by Fermentas; 2 – pBSK plasmid DNA (control); 3 – NucSma(H89G) without hydroxylamine; 4 – NucSma(H89G) with hydroxylamine; 5 – NucSma(wt) without hydroxylamine; 6 – NucSma(wt) with hydroxylamine.

References

  1. Leshchinskaya I.B., Belyaeva M.I., Gil'fanova K.Z. Comparative study of bacterial phosphomono- and phosphodiesterases hydrolyzing nucleic acids and nucleotides. Mikrobiologiya, 1968, vol. 37, pp. 979–983. (In Russian)
  2. Gimadutdinow O.A., Antsilevich L.M. The synthesis of Serratia marcescens extracellular endonuclease by Escherichia coli recombinant strains. Biotekhnologiya, 1993, no. 5, pp. 15–18. (In Russian)
  3. Friedhoff P., Gimadutdinow O., Ruter T., Wende W., Urbanke C., Thole H., Pingoud A. A procedure for renaturation and purification of the extracellular Serratia marcescens nuclease from genetically engineered Escherichia coli. Protein Expression Purif., 1994, vol. 5, no. 1, pp. 37–43.
  4. Friedhoff P., Gimadutdinow O., Pingoud A. Identification of catalytically relevant amino acids of the extracellular Serratia marcescens endonuclease by alignment-guided mutagenesis. Nucleic Acids Res., 1994, vol. 22, no. 16, pp. 3280–3287. (In Russian)
  5. Friedhoff P., Kolmes B., Gimadutdinow O., Wende W., Krause K.L., Pingoud A. Analysis of the mechanism of the Serratia nuclease using site-directed mutagenesis. Nucleic Acids Res., 1996, vol. 24, no. 14, pp. 2632–2639.
  6. Venkataraman P., Lamb R.A., Pinto L.H. Chemical rescue of histidine selectivity filter mutants of the M2 ion channel of influenza A virus. J. Biol. Chem., 2005, vol. 280, no. 22, pp. 21463–21472.
  7. Peracchi A. How (and why) to revive a dead enzyme: The power of chemical rescue. Curr. Chem. Biol., 2008, vol. 2, no. 1, pp. 32–49.
  8. Chen W.J., Lai P.J, Lai Y.S., Huang P.T., Lin C.C., Liao T.H. Probing the catalytic mechanism of bovine pancreatic deoxyribonuclease I by chemical rescue. Biochem. Biophys. Res. Commun., 2007, vol. 352, no. 3, pp. 689–696. doi: 10.1016/j.bbrc.2006.11.078.
  9. Midon M., Gimadutdinow O., Meiss G., Friedhoff P., Pingoud A. Chemical rescue of active site mutants of S. pneumoniae surface endonuclease EndA and other nucleases of the HNH family by imidazole. ChemBioChem, 2012, vol. 13, no. 5, pp. 713–721. doi: 10.1002/cbic.201100775.
  10. Meiss G., Gimadutdinow O., Friedhoff P., Pingoud A. Microtiter-plate assay and related assays for nonspecific endonucleases. Methods Mol. Biol., 2001, vol. 160, pp. 37–48. doi: 10.1385/1-59259-233-3:037.

For citation: Khamidullina R.G., Fazleyeva I.I., Gimadutdinow O.A. Reactivation of Serratia marcescens endonuclease NucSma(H89G) by hydroxilamine. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2017, vol. 159, no. 2, pp. 272–282. (In Russian)


The content is available under the license Creative Commons Attribution 4.0 License.