2.1 Å structure of Serratia endonuclease suggests a mechanism for binding to double-stranded DNA

Miller, Mitchell D.; Tanner, J.J.; Alpaugh, Mary L.; Benedik, Michael J.; Krause, Kurt L.

doi:10.1038/nsb0794-461

Cited by 95 publications

(100 citation statements)

References 24 publications

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“…We also looked for changes in tryptophan fluorescence using the stopped-flow technique, as the nuclease has six tryptophan residues, one of which is conserved among several other homologous nucleases and is located directly within the putative active site (Miller et al, 1994). Two other tryptophan residues are on the side of the enzyme which is believed to bind the substrate.…”

Section: Resultsmentioning

confidence: 99%

“…B., unpublished). In spite of the fact that the primary (Ball et al, 1987), secondary (Friedhoff et al, 1994a), tertiary (Miller et al, 1994) and quaternary structures (Filimonova et al, 1981 ;Friedhoff et al, 199413;Miller and Krause, 1996) of the Serratia nuclease are known, its mechanism of action is not understood. The crystal structure analysis of the Serratia nuclease (Miller et al, 1994), in conjunction with a site-directed mutagenesis study of residues con-served in the Serratia nuclease and related enzymes (Friedhoff et al, 1994a), suggested that His89 [numbering according to the sequence of the mature protein (Ball et al, 19921 and Glu127 among other residues are involved in catalysis; their role, however, is not clear.…”

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confidence: 99%

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Kinetic Analysis of the Cleavage of Natural and Synthetic Substrates by the Serratia Nuclease

Friedhoff

Meiß

Kolmes

et al. 1996

European Journal of Biochemistry

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The extracellular nuclease from Serratia marcescens is a non-specific endonuclease that hydrolyzes double-stranded and single-stranded DNA and RNA with high specific activity. Steady-state and presteady-state kinetic cleavage experiments were performed with natural and synthetic DNA and RNA substrates to understand the mechanism of action of the Serrutia nuclease. Most of the natural substrates are cleaved with similar k,,, and K,,, values, the k,,,lK,,, ratios being comparable to that of staphylococcal nuclease. Substrates with extreme structural features, like poly(dA) . poly(dT) or poly(dG) . poly(dC), are cleaved by the Serratia nuclease with a 50 times higher or 10 times lower K,,,, respectively, as salmon testis DNA. Neither with natural DNA or RNA nor synthetic oligodeoxynucleotide substrates did we observe substrate inhibition for the Serratia nuclease as reported recently. Experiments with short oligodeoxynucleotides confirmed previous results that for moderately good cleavage activity the substrate should contain at least five phosphate residues. Shorter substrates are still cleaved by the Serratia nuclease, albeit at a rate reduced by a factor of more than 100. Cleavage experiments with oligodeoxynucleotides substituted by a single phosphorothioate group showed that the negative charge of the pro-R,-oxygen of the phosphate group 3' adjacent to the scissile phosphodiester bond is essential for cleavage, as only the R,-phosphorothioate supports cleavage at the 5' adjacent phosphodiester bond. Furthermore, the modified bond itself is only cleaved in the R,-diastereomer, albeit 1000 times more slowly than the corresponding unmodified phosphodiester bond, which offers the possibility to determine the stereochemical outcome of cleavage. Pre-steady-state cleavage experiments demonstrate that it is not dissociation of products but association of enzyme and substrate or the cleavage of the phosphodiester bond that is the rate-limiting step of the reaction. Finally, it is shown that Serratia nuclease accepts thymidine 3',5'-bis(p4trophenyl)phosphate as a substrate and cleaves it at its 5'-end to produce nitrophenol and thymidine 3'-@-nitrophenylphosphate) 5-phosphate. The rate of cleavage of this artificial substrate, however, is 6-7 orders of magnitude smaller than the rate of cleavage of macromolecular DNA or RNA.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Kinetic Analysis of the Cleavage of Natural and Synthetic Substrates by the Serratia Nuclease

Friedhoff

Meiß

Kolmes

et al. 1996

European Journal of Biochemistry

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“…[17][18][19][20][21][22]27,32,23 The ␤␤␣-metal binding motif is characterized by one ␣-helix and an antiparallel ␤-sheet, which are separated from each other by a variable length of intervening sequences from 9 to 36. This motif usually binds to divalent metal ions like Zn or Mg.…”

Section: Results and Discussion The ␤␤␣-Metal Motifmentioning

confidence: 99%

“…Table II lists the top ranking hits of proteins and the corresponding structure-aligned sequences. We list only the representative proteins of the protein families, which are colicin E9 (ColE9), 34 the periplasmic nuclease Vvn, 22 the HNH homing endonuclease I-HmuI, 35 the His-Cys box endonuclease I-PpoI, 18 the endonucleases such as Serratia nuclease, 17 CAD/DFF40, 32 the T4 Endo VII, 20 and HIV integrase (IN). 36 The lengths of the intervening sequence between the two ␤-strands vary from 2 to 39.…”

Section: Results and Discussion The ␤␤␣-Metal Motifmentioning

confidence: 99%

“…The motif sequences are usually much less conserved than those of the first type, and the RMS distances of the motif residues from the consensus template are more widely distributed. Examples are the ␤␤␣-metal binding motif, [17][18][19][20][21][22][23] the treble clef finger motif, 24 and the helix-turn-helix motif. 25 Figure 1 shows a typical ␤␤␣-metal binding motif.…”

Section: Introductionmentioning

confidence: 99%

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The fragment transformation method to detect the protein structural motifs

Lin

Chen

et al. 2006

Proteins

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To identify functional structural motifs from protein structures of unknown function becomes increasingly important in recent years due to the progress of the structural genomics initiatives. Although certain structural patterns such as the Asp-His-Ser catalytic triad are easy to detect because of their conserved residues and stringently constrained geometry, it is usually more challenging to detect a general structural motifs like, for example, the ␤␤␣-metal binding motif, which has a much more variable conformation and sequence. At present, the identification of these motifs usually relies on manual procedures based on different structure and sequence analysis tools. In this study, we develop a structural alignment algorithm combining both structural and sequence information to identify the local structure motifs. We applied our method to the following examples: the ␤␤␣-metal binding motif and the treble clef motif. The ␤␤␣-metal binding motif plays an important role in nonspecific DNA interactions and cleavage in host defense and apoptosis. The treble clef motif is a zinc-binding motif adaptable to diverse functions such as the binding of nucleic acid and hydrolysis of phosphodiester bonds. Our results are encouraging, indicating that we can effectively identify these structural motifs in an automatic fashion. Our method may provide a useful means for automatic functional annotation through detecting structural motifs associated with particular functions. Proteins 2006;63:636 -643.

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Simulation of electrostatic and hydrodynamic properties ofSerratia endonuclease

et al. 1997

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We analyze the electrostatic and hydrodynamic properties of a nuclease from the pathogenic gram‐negative bacterium Serratia marcescens using finite‐difference Poisson‐Boltzmann methods for electrostatic calculations and a bead‐model approach for diffusion coefficient calculations. Electrostatic properties are analyzed for the enzyme in monomeric and dimeric forms and also in the context of DNA binding by the nuclease. Our preliminary results show that binding of a double‐stranded DNA dodecamer by nuclease causes an overall shift in the charge of the protein by approximately three units of elementary charge per monomer, resulting in a positively charged protein at physiologic pH. In these calculations, the free enzyme was found to have a negative (−1 e) charge per monomer at pH 7. The most dramatic shift in pKa involves His 89 whose pKa increases by three pH units upon DNA binding. This shift leads to a protonated residue at pH 7, in contrast to the unprotonated form in the free enzyme. DNA binding also leads to a decrease in the energetic distances between the most stable protonation states of the enzyme. Dimerization has no significant effect on the electrostatic properties of each of the monomers for both free enzyme and that bound to DNA. Results of hydrodynamic calculations are consistent with the dimeric form of the enzyme in solution. The computed translational diffusion coefficient for the dimer model of the enzyme is in very good agreement with measurements from light scattering experiments. Preliminary electrooptical calculations indicate that the dimer should possess a large dipole moment (approximately 600 Debye units) as well as substantial optical anisotropy (limiting reduced linear electric dichroism of about 0.3). Therefore, this system may serve as a good model for investigation of electric and hydrodynamic properties by relaxation electrooptical experiments. © 1997 John Wiley & Sons, Inc.

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2.1 Å structure of Serratia endonuclease suggests a mechanism for binding to double-stranded DNA

Cited by 95 publications

References 24 publications

Kinetic Analysis of the Cleavage of Natural and Synthetic Substrates by the Serratia Nuclease

Kinetic Analysis of the Cleavage of Natural and Synthetic Substrates by the Serratia Nuclease

The fragment transformation method to detect the protein structural motifs

Simulation of electrostatic and hydrodynamic properties ofSerratia endonuclease

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