Analysis of the reaction mechanism of the non‐specific endonuclease of <i>Serratia marcescens</i> using an artificial minimal substrate

Kolmes, Bettina; Franke, Ingo; Friedhoff, Peter; Pingoud, Alfred

doi:10.1016/s0014-5793(96)01210-0

Cited by 29 publications

(43 citation statements)

References 19 publications

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“…3. Interestingly, with Serratia nuclease, the presence of phosphate 3P to the bond to be cleaved is essential for hydrolysis [133,134]. Similar observations were made in the case of Anabaena nuclease [119].…”

Section: Substrate Speci¢citysupporting

confidence: 63%

Sugar non-specific endonucleases

Rangarajan¹

2001

FEMS Microbiology Reviews

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Sugar non-specific endonucleases are multifunctional enzymes and are widespread in distribution. Apart from nutrition, they have also been implicated in cellular functions like replication, recombination and repair. Their ability to recognize different DNA structures has also been exploited for the determination of nucleic acid structure. Although more than 30 non-specific endonucleases have been isolated to date, very little information is available regarding their structure^function correlations except that of staphylococcal and Serratia nucleases. However, during the past few years, the primary structure, nature of the active site based on sequence homology, and the probable mechanism of action have been postulated for some of the enzymes. This review describes the purification, characteristics, biological role and applications of sugar non-specific endonucleases. ß

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Section: Substrate Speci¢citysupporting

confidence: 63%

Sugar non-specific endonucleases

Rangarajan¹

2001

FEMS Microbiology Reviews

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“…Glu 127 seems to be involved in protonation of the leaving group. These residues are conserved in the Serratia family of nucleases and their essential function has been demonstrated for Serratia nuclease (29,31,32) as well as for Anabaena nuclease. 3 While it appears from a comparison of the properties of these two nucleases that they have a similar mechanism of action (6), they differ as mentioned above in their quaternary structure.…”

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confidence: 99%

On the Advantage of Being a Dimer, a Case Study Using the DimericSerratia Nuclease and the Monomeric Nuclease fromAnabaena sp. Strain PCC 7120

Franke

Meiß

Pingoud

1999

Journal of Biological Chemistry

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The extracellular endonucleases from Serratia marcescens and Anabaena sp. are members of a family of nonspecific endonucleases. In contrast to the monomeric Anabaena nuclease, the Serratia nuclease is a dimer of two identical subunits. To find out whether the two active sites of the Serratia nuclease function independently of each other and what the advantage of being a dimer for this enzyme might be, we produced (i) dimers in which the two subunits were cross-linked, (ii) heterodimers consisting of a wild type and an inactive mutant subunit which were also cross-linked, and (iii) monomeric variants which are unable to dimerize. The monomeric H184R variant and the cross-linked S140C variant exhibit the same activity as the wild type enzyme, while the cross-linked heterodimer with one inactive subunit shows only half of the activity of the wild type enzyme, demonstrating functional independence of the two subunits of the Serratia nuclease. On the other hand at low enzyme and substrate concentrations dimeric forms of the Serratia nuclease are relatively more active than monomeric forms or the monomeric Anabaena nuclease in cleaving polynucleotides, not, however, oligonucleotides, which is correlated with the ability of dimeric forms of the Serratia nuclease to form large enzyme-substrate networks with high molecular weight DNA and to cleave polynucleotides in a processive manner. We conclude that in the natural habitat of Serratia marcescens where the supply of nutrients may become growth limiting the dimeric nuclease can fulfil its nutritive function more efficiently than a monomeric enzyme.

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“…This residue could have an additional role in positioning the attacking water molecule relative to the phosphorus atom. A similar function has been discussed for R57, acting through its guanidinium group [33,55]. …”

Section: Catalytic Mechanismmentioning

confidence: 67%

“…The chromophoric nuclease substrate deoxythymidine 3P,5P-bis-(p-nitrophenyl-phosphate) however is cleaved di¡erently by DNase I and Serratia nuclease. DNase I cleaves the phosphodiester bond on the 3P side [33], and Serratia nuclease attacks the 5P-side of the ribose sugar moiety of this particular substrate analog. This may re£ect a functional di¡erence in substrate recognition and cleavage by these enzymes or, more likely, may simply suggest some peculiarities of these enzymes' binding to this arti¢cial substrate.…”

Section: A Family Of Nucleasesmentioning

confidence: 99%

“…R87, R131 and possibly also D86 mutants are mainly affected in their ability to bind nucleic acid substrate but not in their catalytic activity [55]. More speci¢-cally, R87 is thought to interact with the phosphate group at the 3P-end of the ribose sugar [33], which is essential for cleavage near the 5P-end [20]. With regard to the nuclease model, all three residues are located in the putative substrate binding site of the enzyme, suitably positioned to assist in positioning diverse nucleotide substrates into a conformation that is accepted by the enzyme [48].…”

Section: Catalytic Mechanismmentioning

confidence: 99%

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Serratia marcescens and its extracellular nuclease

Benedik¹

1998

FEMS Microbiology Letters

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Serratia marcescens produces an endonuclease with extraordinarily high specific activity that is released into the surrounding medium. This enzyme has been the focus of studies on gene regulation, protein secretion, endonuclease action, and protein structure; it has also been found to have many applications in biotechnology. Here we briefly review these different facets of research regarding the Serratia nuclease and summarize the current state of knowledge about this enzyme. z

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Analysis of the reaction mechanism of the non‐specific endonuclease of Serratia marcescens using an artificial minimal substrate

Cited by 29 publications

References 19 publications

Sugar non-specific endonucleases

Sugar non-specific endonucleases

On the Advantage of Being a Dimer, a Case Study Using the DimericSerratia Nuclease and the Monomeric Nuclease fromAnabaena sp. Strain PCC 7120

Serratia marcescens and its extracellular nuclease

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