Genetic engineering, production and characterisation of monomeric variants of the dimeric <i>Serratia marcescens</i> endonuclease

Franke, Ingo; Meiß, Gregor; Blecher, D.; Gimadutdinow, Oleg; Urbanke, Claus; Pingoud, Alfred

doi:10.1016/s0014-5793(98)00279-8

Cited by 22 publications

(24 citation statements)

References 27 publications

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“…3). It had been demonstrated before that the H89A, the H184A, N, T, or R, and the cross-linked S140C variants all have the same CD spectrum as the wild type enzyme indicating that the secondary structure composition and presumably also the tertiary structure is unaltered by the amino acid substitution(s) and the cross-linking (41,45).…”

Section: Resultsmentioning

confidence: 94%

“…Obligatory monomers were obtained by replacing His 184 which is located in the dimer interface of Serratia nuclease (5) by Ala, Thr, Asn, and Arg (45).…”

mentioning

confidence: 99%

“…Analysis of the Quaternary Structure of Serratia and Anabaena Nuclease-The quaternary structure analysis of Serratia nuclease variants and of Anabaena nuclease was carried out using gel filtration and analytical ultracentrifugation (6,28,45).…”

mentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 94%

“…Obligatory monomers were obtained by replacing His 184 which is located in the dimer interface of Serratia nuclease (5) by Ala, Thr, Asn, and Arg (45).…”

mentioning

confidence: 99%

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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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“…His 184 , which forms a hydrogen bond with the Ser 229 and with the Pro 180 carbonyl in the interchain region, corresponds to Arg 222 in NucA. It has previously been noted that the H184R mutant of the Serratia nuclease is monomeric (11). The interstrand salt bridge (Asp 225 -Arg 136 ) in the Serratia structure would not exist in NucA, which has Asn and Gln residues at these positions.…”

Section: Discussionmentioning

confidence: 99%

Structural Insights into the Mechanism of Nuclease A, a ββα Metal Nuclease from Anabaena

Ghosh

Meiß

Pingoud

et al. 2005

Journal of Biological Chemistry

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Nuclease A (NucA) is a nonspecific endonuclease from Anabaena sp. capable of degrading single-and doublestranded DNA and RNA in the presence of divalent metal ions. We have determined the structure of the ⌬(2-24),D121A mutant of NucA in the presence of Zn 2؉and Mn 2؉ (PDB code 1ZM8). The mutations were introduced to remove the N-terminal signal peptide and to reduce the activity of the nonspecific nuclease, thereby reducing its toxicity to the Escherichia coli expression system. NucA contains a ␤␤␣ metal finger motif and a hydrated Mn 2؉ ion at the active site. Unexpectedly, NucA was found to contain additional metal binding sites ϳ26 Å apart from the catalytic metal binding site. A structural comparison between NucA and the closest analog for which structural data exist, the Serratia nuclease, indicates several interesting differences. First, NucA is a monomer rather than a dimer. Second, there is an unexpected structural homology between the N-terminal segments despite a poorly conserved sequence, which in Serratia includes a cysteine bridge thought to play a regulatory role. In addition, although a sequence alignment had suggested that NucA lacks a proposed catalytic residue corresponding to Arg 57 in Serratia, the structure determined here indicates that Arg 93 in NucA is positioned to fulfill this role. Based on comparison with DNA-bound nuclease structures of the ␤␤␣ metal finger nuclease family and available mutational data on NucA, we propose that His 124 acts as a catalytic base, and Arg 93 participates in the catalysis possibly through stabilization of the transition state.

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“…Moreover, the dimeric forms of non-speci¢c endonucleases from S. racemosum [34] and S. marcescens [87] are held together purely by non-covalent interactions and not by disul¢de [86]. Although the monomeric variants were functionally independent, in the presence of low enzyme concentration and high molecular mass DNA the native dimeric form of S. marcescens nuclease was relatively more active than the monomeric form or the heterodimer with one inactive subunit [89].…”

Section: Puri¢cationmentioning

confidence: 98%

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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Genetic engineering, production and characterisation of monomeric variants of the dimeric Serratia marcescens endonuclease

Cited by 22 publications

References 27 publications

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

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

Structural Insights into the Mechanism of Nuclease A, a ββα Metal Nuclease from Anabaena

Sugar non-specific endonucleases

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