Identification of catalytically relevant amino acids of the extracellular<i>Serratia marcescens</i>endonuclease by alignment-guided mutagenesis

Friedhoff, Peter; Gimadutdinow, Oleg; Pingoud, Alfred

doi:10.1093/nar/22.16.3280

Cited by 69 publications

(66 citation statements)

References 32 publications

(26 reference statements)

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“…Both the CJE0566 and CJE1441 protein sequences contained a conserved domain of the NUC superfamily (SMART accession no. SM00477) characteristic for DNA/RNA nonspecific endonucleases (11,12). Moreover, the translated sequences contained a DRGH motif (see Fig.…”

Section: Resultsmentioning

confidence: 99%

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Nucleases Encoded by the Integrated Elements CJIE2 and CJIE4 Inhibit Natural Transformation of Campylobacter jejuni

et al. 2010

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The species Campylobacter jejuni is naturally competent for DNA uptake; nevertheless, nonnaturally transformable strains do exist. For a subset of strains we previously showed that a periplasmic DNase, encoded by dns, inhibits natural transformation in C. jejuni. In the present study, genetic factors coding for DNase activity in the absence of dns were identified. DNA arrays indicated that nonnaturally transformable dns-negative strains contain putative DNA/RNA nonspecific endonucleases encoded by CJE0566 and CJE1441 of strain RM1221. These genes are located on C. jejuni integrated elements 2 and 4. Expression of CJE0566 and CJE1441 from strain RM1221 and a homologous gene from strain 07479 in DNase-negative Escherichia coli and C. jejuni strains indicated that these genes code for DNases. Genetic transfer of the genes to a naturally transformable C. jejuni strain resulted in a decreased efficiency of natural transformation. Modeling suggests that the C. jejuni DNases belong to the Serratia nuclease family. Overall, the data indicate that the acquisition of prophageencoded DNA/RNA nonspecific endonucleases inhibits the natural transformability of C. jejuni through hydrolysis of DNA.

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Section: Resultsmentioning

confidence: 99%

“…PDOC00821} in which histidine is the active site residue (11,12). Although the amino acid sequences and the predicted 3D structures of CJE0566 and CJE1441 share similarity with members of this family, the sequences deviate from the DRGH motif in their fifth residue (Q3T).…”

Section: Discussionmentioning

confidence: 99%

Nucleases Encoded by the Integrated Elements CJIE2 and CJIE4 Inhibit Natural Transformation of Campylobacter jejuni

et al. 2010

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“…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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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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“…The extracellular nuclease (NucA) is the major focus of this study. This nuclease has been well characterized biochemically and structurally (15,37), but little is known about its biosynthesis or export. Here we show that the secretion of this nuclease occurs in two steps.…”

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Two-step secretion of the Serratia marcescens extracellular nuclease

et al. 1996

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The extracellular nuclease of Serratia marcescens is one of a wide variety of enzymes secreted into the growth medium. Its appearance occurs late in the growth of a culture, and its gene, nucA, is transcriptionally regulated in a complex fashion by growth phase and other factors. Pulse-labeling studies reveal that extracellular secretion of nuclease occurs as a two-step process. In the first step, nuclease is rapidly translocated across the cytoplasmic membrane into the periplasm, where it accumulates as a mature active nuclease. A precursor protein, nuclease still carrying its signal sequence, was detected in the presence of carbonyl cyanide m-chlorophenylhydrazone or sodium azide, suggesting that this initial translocation and signal processing step involves an energy-dependent and Sec-dependent pathway in S. marcescens. The second step of secretion across the outer membrane is a slow process requiring between 30 to 120 min, depending on growth conditions.Gram-negative bacteria use diverse mechanisms for the secretion of extracellular proteins (33,44,47,54). Proteins with N-terminal signal sequences often utilize the general secretory pathway (GSP) (44). The GSP consists of a two-step process in which the first step is initiated by the signal sequence and its interaction with the Sec proteins to translocate the protein across the cytoplasmic membrane to the periplasm (48). The second step, which involves translocation across the outer membrane to the extracellular medium, requires an accessory secretion apparatus encoded by numerous genes. One of the best-characterized proteins using this system, the pullulanase of Klebsiella oxytoca, requires 14 gene products for the second secretion step (44). Components of this secretion pathway are well conserved among gram-negative bacteria, such as Pseudomonas aeruginosa (13, 53), Xanthomonas campestris (10, 22), Aeromonas hydrophila (25), Erwinia chrysanthemi (18, 32), and E. carotovora (46). However other extracellular proteins are known to utilize mechanisms which differ from that of the GSP, such as the type III secretion systems (47) and the ABC transporter systems (54) as well as some unique systems (38,49).Serratia marcescens secretes a variety of enzymes into the extracellular milieu, including a nuclease (3, 11), a phospholipase A (17), a lipase (19), two chitinases (27, 39), proteases (7,40), and an iron chelator, HasA (30), and it has multiple mechanisms to secrete them. As such, it provides a good model with which to study different pathways of secretion. The extracellular HasA, the metalloprotease, and the lipase of S. marcescens utilize ABC transporter systems, a signal sequenceindependent secretion pathway analogous to that used for secretion of ␣-hemolysin in Escherichia coli (1, 31, 51). The serine protease from S. marcescens is a self-secreting protein that utilizes an N-terminal signal sequence to cross the inner membrane but mediates its own translocation across the outer membrane by using its carboxy-terminal domain (38). A hemolysin encoded by the s...

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Identification of catalytically relevant amino acids of the extracellularSerratia marcescensendonuclease by alignment-guided mutagenesis

Cited by 69 publications

References 32 publications

Nucleases Encoded by the Integrated Elements CJIE2 and CJIE4 Inhibit Natural Transformation of Campylobacter jejuni

Nucleases Encoded by the Integrated Elements CJIE2 and CJIE4 Inhibit Natural Transformation of Campylobacter jejuni

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

Two-step secretion of the Serratia marcescens extracellular nuclease

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