Search tips
Search criteria 


Logo of narLink to Publisher's site
Nucleic Acids Res. 1994 August 25; 22(16): 3280–3287.
PMCID: PMC523719

Identification of catalytically relevant amino acids of the extracellular Serratia marcescens endonuclease by alignment-guided mutagenesis.


By sequence alignment of the extracellular Serratia marcescens nuclease with three related nucleases we have identified seven charged amino acid residues which are conserved in all four sequences. Six of these residues together with four other partially conserved His or Asp residues were changed to alanine by site-directed PCR-mediated mutagenesis using a variant of the nuclease gene in which the coding sequence of the signal peptide was replaced by the coding sequence for an N-terminal affinity tag [Met(His)6GlySer]. Four of the mutant proteins showed almost no reduction in nuclease activity but five displayed a 10- to 1000-fold reduction in activity and one (His110Ala) was inactive. Based upon these results it is suggested that the S.marcescens nuclease employs a mechanism in which His110 acts in concert with a Mg2+ ion and three carboxylates (Asp107, Glu148 and Glu232) as well as one or two basic amino acid residues (Arg108, Arg152).

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.6M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Nestle M, Roberts WK. An extracellular nuclease from Serratia marcescens. II. Specificity of the enzyme. J Biol Chem. 1969 Oct 10;244(19):5219–5225. [PubMed]
  • Ball TK, Suh Y, Benedik MJ. Disulfide bonds are required for Serratia marcescens nuclease activity. Nucleic Acids Res. 1992 Oct 11;20(19):4971–4974. [PMC free article] [PubMed]
  • Ball TK, Saurugger PN, Benedik MJ. The extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli. Gene. 1987;57(2-3):183–192. [PubMed]
  • Biedermann K, Jepsen PK, Riise E, Svendsen I. Purification and characterization of a Serratia marcescens nuclease produced by Escherichia coli. Carlsberg Res Commun. 1989;54(1):17–27. [PubMed]
  • Filimonova MN, Baratova LA, Vospel'nikova ND, Zheltova AO, Leshchinskaia IB. Endonukleaza Serratia marcescens. Kharakteristika fermenta. Biokhimiia. 1981 Sep;46(9):1660–1666. [PubMed]
  • Yonemura K, Matsumoto K, Maeda H. Isolation and characterization of nucleases from a clinical isolate of Serratia marcescens kums 3958. J Biochem. 1983 May;93(5):1287–1295. [PubMed]
  • Bannikova GE, Blagova EV, Dementiev AA, Morgunova EYu, Mikchailov AM, Shlyapnikov SV, Varlamov VP, Vainshtein BK. Two isoforms of Serratia marcescens nuclease. Crystallization and preliminary X-ray investigation of the enzyme. Biochem Int. 1991 Jul;24(5):813–822. [PubMed]
  • Pedersen J, Filimonova M, Roepstorff P, Biedermann K. Characterization of Serratia marcescens nuclease isoforms by plasma desorption mass spectrometry. Biochim Biophys Acta. 1993 Sep 3;1202(1):13–21. [PubMed]
  • Friedhoff P, Gimadutdinow O, Rüter 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 Expr Purif. 1994 Feb;5(1):37–43. [PubMed]
  • Miller MD, Benedik MJ, Sullivan MC, Shipley NS, Krause KL. Crystallization and preliminary crystallographic analysis of a novel nuclease from Serratia marcescens. J Mol Biol. 1991 Nov 5;222(1):27–30. [PubMed]
  • Muro-Pastor AM, Flores E, Herrero A, Wolk CP. Identification, genetic analysis and characterization of a sugar-non-specific nuclease from the cyanobacterium Anabaena sp. PCC 7120. Mol Microbiol. 1992 Oct;6(20):3021–3030. [PubMed]
  • Zabeau M, Stanley KK. Enhanced expression of cro-beta-galactosidase fusion proteins under the control of the PR promoter of bacteriophage lambda. EMBO J. 1982;1(10):1217–1224. [PubMed]
  • Courtney M, Buchwalder A, Tessier LH, Jaye M, Benavente A, Balland A, Kohli V, Lathe R, Tolstoshev P, Lecocq JP. High-level production of biologically active human alpha 1-antitrypsin in Escherichia coli. Proc Natl Acad Sci U S A. 1984 Feb;81(3):669–673. [PubMed]
  • Thielking V, Selent U, Köhler E, Wolfes H, Pieper U, Geiger R, Urbanke C, Winkler FK, Pingoud A. Site-directed mutagenesis studies with EcoRV restriction endonuclease to identify regions involved in recognition and catalysis. Biochemistry. 1991 Jul 2;30(26):6416–6422. [PubMed]
  • Sinha ND, Biernat J, McManus J, Köster H. Polymer support oligonucleotide synthesis XVIII: use of beta-cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucleic Acids Res. 1984 Jun 11;12(11):4539–4557. [PMC free article] [PubMed]
  • Schuler GD, Altschul SF, Lipman DJ. A workbench for multiple alignment construction and analysis. Proteins. 1991;9(3):180–190. [PubMed]
  • Rost B, Sander C. Prediction of protein secondary structure at better than 70% accuracy. J Mol Biol. 1993 Jul 20;232(2):584–599. [PubMed]
  • Ito W, Ishiguro H, Kurosawa Y. A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction. Gene. 1991 Jun 15;102(1):67–70. [PubMed]
  • Mach H, Middaugh CR, Lewis RV. Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins. Anal Biochem. 1992 Jan;200(1):74–80. [PubMed]
  • KUNITZ M. Crystalline desoxyribonuclease; isolation and general properties; spectrophotometric method for the measurement of desoxyribonuclease activity. J Gen Physiol. 1950 Mar;33(4):349–362. [PMC free article] [PubMed]
  • Deléage G, Geourjon C. An interactive graphic program for calculating the secondary structure content of proteins from circular dichroism spectrum. Comput Appl Biosci. 1993 Apr;9(2):197–199. [PubMed]
  • Chen YH, Yang JT, Chau KH. Determination of the helix and beta form of proteins in aqueous solution by circular dichroism. Biochemistry. 1974 Jul 30;13(16):3350–3359. [PubMed]
  • Yanofsky SD, Love R, McClarin JA, Rosenberg JM, Boyer HW, Greene PJ. Clustering of null mutations in the EcoRI endonuclease. Proteins. 1987;2(4):273–282. [PubMed]
  • Dorner LF, Schildkraut I. Direct selection of binding proficient/catalytic deficient variants of BamHI endonuclease. Nucleic Acids Res. 1994 Mar 25;22(6):1068–1074. [PMC free article] [PubMed]
  • Lin JJ, Sancar A. Active site of (A)BC excinuclease. I. Evidence for 5' incision by UvrC through a catalytic site involving Asp399, Asp438, Asp466, and His538 residues. J Biol Chem. 1992 Sep 5;267(25):17688–17692. [PubMed]
  • Doherty AJ, Worrall AF, Connolly BA. Mutagenesis of the DNA binding residues in bovine pancreatic DNase 1: an investigation into the mechanism of sequence discrimination by a sequence selective nuclease. Nucleic Acids Res. 1991 Nov 25;19(22):6129–6132. [PMC free article] [PubMed]
  • Weston SA, Lahm A, Suck D. X-ray structure of the DNase I-d(GGTATACC)2 complex at 2.3 A resolution. J Mol Biol. 1992 Aug 20;226(4):1237–1256. [PubMed]
  • Beese LS, Steitz TA. Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J. 1991 Jan;10(1):25–33. [PubMed]
  • Yang W, Hendrickson WA, Crouch RJ, Satow Y. Structure of ribonuclease H phased at 2 A resolution by MAD analysis of the selenomethionyl protein. Science. 1990 Sep 21;249(4975):1398–1405. [PubMed]
  • Katayanagi K, Okumura M, Morikawa K. Crystal structure of Escherichia coli RNase HI in complex with Mg2+ at 2.8 A resolution: proof for a single Mg(2+)-binding site. Proteins. 1993 Dec;17(4):337–346. [PubMed]
  • Davies JF, 2nd, Hostomska Z, Hostomsky Z, Jordan SR, Matthews DA. Crystal structure of the ribonuclease H domain of HIV-1 reverse transcriptase. Science. 1991 Apr 5;252(5002):88–95. [PubMed]
  • Jeltsch A, Alves J, Wolfes H, Maass G, Pingoud A. Substrate-assisted catalysis in the cleavage of DNA by the EcoRI and EcoRV restriction enzymes. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8499–8503. [PubMed]
  • Jeltsch A, Alves J, Maass G, Pingoud A. On the catalytic mechanism of EcoRI and EcoRV. A detailed proposal based on biochemical results, structural data and molecular modelling. FEBS Lett. 1992 Jun 8;304(1):4–8. [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press