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A Pingoud

Publications and source records attributed to A Pingoud.

At least 37 records · Page 2Linked to original sources
PubMed1999

Synthesis and biochemical characterization of obligatory dimers of the sugar non-specific nuclease from Serratia marcescens using specifically designed bismaleimidoalkanes as SH-specific crosslinking reagents.

The genetically engineered S140C variant of the homodimeric nuclease from Serratia marcescens was crosslinked across the dimer interface at the Cys 140 residues using bifunctional SH-specific 1,1'-alkanediyl-bis-pyrrole-2,5-diones of different lengths. These bismaleimidoalkanes were synthesized by the condensation of n-alkyldiamines with maleic anhydride and subsequent cyclization with acetic anhydride and sodium acetate. Bismaleimidohexane (BMH) which gave the best crosslinking yield was used to produce in preparative amounts crosslinked Serratia nuclease. The crosslinked protein has the same secondary structure and exhibits the same guanidinium chloride unfolding behavior as the wild type enzyme or the non-covalently linked S 140C variant. In contrast, in thermal unfolding experiments the crosslinked dimer behaves differently from the wild type enzyme or the non-covalently linked S140C variant. CD-spectra recorded during temperature rise showed only minor changes of the secondary structure composition for the wild type enzyme and the non-covalently linked S140C variant, whereas in the case of the crosslinked S140C dimer a distinct increase of the CD effect was observed corresponding to an increase in alpha-helix. Our results demonstrate that bismaleimidoalkanes are very well suited to covalently link subunits of proteins, provided suitably located cysteine residues are present.

Circular Dichroism↗
PubMed1999

Application of oligonucleoside methylphosphonates in the studies on phosphodiester hydrolysis by Serratia endonuclease.

The endonuclease from Serratia marcescens is a non-specific enzyme that cleaves single and double stranded RNA and DNA. It accepts a phosphorylated pentanucleotide as a minimal substrate which is cleaved in the presence of Mg2+ at the second phosphodiester linkage. The present study is aimed at understanding the role of electrostatic and hydrogen bond interactions in phosphodiester hydrolysis. Towards this objective, six pentadeoxyadenylates with single stereoregular methylphosphonate substitution within this minimal substrate (2a-4b) were synthesized following a protocol described here. These modified oligonucleotides were used as substrates for the Serratia nuclease. The enzyme interaction studies revealed that the enzyme failed to hydrolyze any of the methylphosphonate analogues suggesting the importance of negative charge and/or hydrogen bond acceptors in binding and cleavage of its substrate. Based on these results and available site-directed mutagenesis as well as structural data, a model for nucleic acid binding by Serratia nuclease is proposed.

Binding Sites↗
PubMed1999

Defining the location and function of domains of McrB by deletion mutagenesis.

The GTP-dependent restriction endonuclease McrBC of E. coli K12, which recognizes cytosine-methylated DNA, consists of two protein subunits, McrB and McrC. We have investigated the structural assignment and interdependence of the McrB subunit functions, namely (i) specific DNA recognition and (ii) GTP binding and hydrolysis. Extending earlier work, we have produced McrB variants comprising N- and C-terminal fragments. The variants McrB1-162 and McrB1-170 are still capable of specific DNA binding. McrB169-465 shows GTP binding and hydrolysis characteristics indistinguishable from full-length McrB as well as wild-type like interaction with McrC. Thus, DNA and GTP binding are spatially separated on the McrB molecule, and the respective domains function quite independently.

Amino Acid Sequence↗
PubMed1998

Crosslinking the EcoRV restriction endonuclease across the DNA-binding site reveals transient intermediates and conformational changes of the enzyme during DNA binding and catalytic turnover.

EcoRV completely encircles bound DNA with two loops, forming the entry and exit gate for the DNA substrate. These loops were crosslinked generating CL-EcoRV which binds and releases linear DNA only slowly, because threading linear DNA into and out of the DNA-binding 'tunnel' of CL-EcoRV is not very effective. If the crosslinking reaction is carried out with a circular bound DNA, CL-EcoRV is hyperactive towards the trapped substrate which is cleaved very quickly but not very accurately. CL-EcoRV also binds to, but does not cleave, circular DNA when added from the outside, because it cannot enter the active site. Based on these results a two-step binding model is proposed for EcoRV: initial DNA binding occurs at the outer side of the loops before the gate opens and then the DNA is transferred to the catalytic center.

Amino Acid Substitution↗
PubMed1998

Protein engineering of the restriction endonuclease EcoRV--structure-guided design of enzyme variants that recognize the base pairs flanking the recognition site.

We generated variants of the restriction endonuclease EcoRV that discriminate between recognition sites with different flanking sequences. This was achieved by designing new contacts to the bases in the major groove of the DNA preceding and following the EcoRV recognition site. We selected Ala181 as the starting point for the extension of the site specificity of EcoRV because, according to the structure of the specific EcoRV x DNA complex, this residue is involved in a water mediated contact with the bases flanking the recognition sequence on the 5' side. A substitution of this alanine residue by other amino acid residues changes the protein-DNA interface in this region and potentially creates new contacts, such that EcoRV variants could have an extended specificity, i.e. a greater selectivity for EcoRV sites within a particular sequence context. EcoRV variants with naturally occurring amino acid residues at position 181 were produced and their selectivity analyzed with oligodeoxynucleotide and plasmid substrates that differ only in the base pairs immediately flanking the EcoRV site. Some variants, having amino acid residues with long or bulky side chains at position 181 showed altered preferences for the base pairs flanking the recognition sequence with oligodeoxynucleotide substrates without loosing their catalytic efficiency. One variant, A181K, is able to discriminate between purine and pyrimidine bases on the 5' side of the recognition sequence, probably by means of a new hydrogen bond to the N7 of the purine base. Another variant, A181E, strongly prefers a thymine base on the 5' side of the recognition sequence, presumably due to a hydrogen bond formed between the protonated glutamic acid residue and the O4 of thymine.

Base Sequence↗
PubMed1998

Structural and functional analysis of the homing endonuclease PI-sceI by limited proteolytic cleavage and molecular cloning of partial digestion products.

PI-SceI is a member of an unusual class of rare cutting homing endonucleases produced by an autocatalytic protein splicing from a precursor. To analyze the structural and functional domain organization of the endonuclease PI-SceI and to examine whether the DNA binding activity can be structurally separated from the catalytic activity, we performed limited proteolytic digestion experiments with various proteases. Two protease-resistant fragments spanning the N- and C-terminal halves of the nuclease were identified using different proteases which cleave the protein in the same region. Each fragment contains one of the two conserved LAGLIDADG motifs. The products of the limited proteolytic digests were shown to remain associated and to exhibit specific DNA binding but to be inactive in DNA cleavage. Different from what is observed with native PI-SceI, only one complex is formed as shown in an electrophoretic mobility shift assay. Expression clones for the N- and C-terminal protein fragments obtained by tryptic digestion were constructed, and the proteins PI-SceI-N and PI-SceI-C were purified. Only PI-SceI-N exhibits DNA binding activity. Bending experiments with PI-SceI-N, a mixture of PI-SceI-N and PI-SceI-C, as well as the products of the limited tryptic digest show that a DNA substrate with the full length recognition sequence is bent by 45;. This degree of bending is also observed with a DNA containing only the right side of the recognition sequence, corresponding to one of the DNA cleavage products of PI-SceI. Our results demonstrate that the N-terminal half of PI-SceI which lacks one of the two LAGLDADG motifs is able to bind to DNA specifically and to induce one of the distortions observed to occur in the process of DNA binding by PI-SceI. These results are discussed in light of the recently solved crystal structure of PI-SceI and used to refine a model for the mechanism of DNA binding and cleavage by PI-SceI.

Amino Acid Sequence↗
PubMed1998

Intra- vs intersubunit communication in the homodimeric restriction enzyme EcoRV: Thr 37 and Lys 38 involved in indirect readout are only important for the catalytic activity of their own subunit.

EcoRV is a dimer of two identical subunits which together form one binding site for the double-stranded DNA substrate. Concerted cleavage of both strands of the duplex requires intersubunit communication to synchronize the two catalytic centers of EcoRV. Here we address the question of how contacts to the DNA backbone trigger conformational changes which lead to the activation of both catalytic centers. The structure of the specific EcoRV-DNA complex shows that a region including amino acids Thr 37 and Lys 38 is involved in interactions with the DNA backbone and is a candidate for intersubunit communication. Homodimeric EcoRV T37A and K38A variants have a 1000-fold reduced catalytic activity. To examine whether Thr 37 and Lys 38 of one subunit affect the catalytic center in the same subunit and/or in the other subunit, we have produced heterodimeric variants containing a Thr 37 --> Ala or Lys 38 --> Ala substitution in one subunit combined with a wild type (wt) subunit (wt/T37A and wt/K38A) or with a subunit which contains an amino acid substitution (Asp 90 --> Ala) in the active site (D90A/T37A and D90A/K38A). Cleavage experiments with supercoiled pAT153 show that wt/T37A and wt/K38A preferentially nick the DNA. A steady-state kinetic analysis of the cleavage of an oligodeoxynucleotide substrate shows that the activity of wt/T37A and wt/K38A is half of that of wild type EcoRV, whereas D90A/T37A and D90A/K38A are almost inactive. These results demonstrate that Thr 37 and Lys 38 affect primarily the catalytic center in their own subunit and that both subunits of EcoRV can be activated independently of each other. We suggest that Thr 37 and Lys 38 control the catalytic activity of the active site in their own subunit by positioning alpha-helix B.

Catalysis↗
PubMed1998

The protein splicing domain of the homing endonuclease PI-sceI is responsible for specific DNA binding.

The homing endonuclease PI- Sce I consists of a protein splicing domain (I) and an endonucleolytic domain (II). To characterize the two domains with respect to their contribution to DNA recognition we cloned, purified and characterized the isolated domains. Both domains have no detectable endonucleolytic activity. Domain I binds specifically to the PI- Sce I recognition sequence, whereas domain II displays only weak non-specific DNA binding. In the specific complex with domain I the DNA is bent to a similar extent as observed with the initial complex formed between PI- Sce I and DNA. Our results indicate that protein splicing domain I is also involved in recognition of the DNA substrate.

Amino Acid Sequence↗
PubMed1998

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

The Serratia nuclease is a non-specific endonuclease which cleaves single- and double-stranded RNA and DNA. It is a member of a large family of related endonucleases, most of which are dimers of identical subunits, with the notable exception of the Anabaena nuclease which is a monomer. In order to find out whether the dimer state of the Serratia nuclease is essential for its function we have produced variants of this nuclease which based on the crystal structure (Miller, M.D. and Krause, K.L. (1996), Protein Science 5, 24-33) were expected to be unable to dimerise. We demonstrate here that these variants, H184A, H184N, H184T and H184R, are monomers and have the same secondary structure, stability towards chemical denaturation and activity as the wild-type enzyme. This allows to conclude that the dimeric state is not essential for the catalytic function of the Serratia nuclease. In contrast, the S179C variant which is also a monomer shows little activity, presumably because this amino acid substitution changes the structure of the enzyme.

Bacterial Proteins↗
PubMed1998

Engineering of variants of the restriction endonuclease EcoRV that depend in their cleavage activity on the flexibility of sequences flanking the recognition site.

The present work describes mutants of the restriction enzyme EcoRV that discriminate very efficiently between oligodeoxynucleotide substrates with an EcoRV recognition sequence in different sequence context. All of these EcoRV variants harbor substitutions at position 226, where in the cocrystal structure of the specific EcoRV/DNA complex an arginine contacts the backbone of the DNA substrate upstream of the recognition sequence, and cleave an oligodeoxynucleotide with an EcoRV site in a nonflexible sequence context (the recognition site being flanked by runs of A and T) with much higher catalytic efficiency (kcat/Km) than an oligodeoxynucleotide with an EcoRV site in a flexible sequence context (the recognition site being flanked by runs of AT), in contrast to the wild-type enzyme, that cleaves both substrates with the same catalytic efficiency. Steady-state and single-turnover kinetics indicate that the enhanced selectivity of the mutants is due to the catalytic step of the reaction. It is possible to enhance the discriminatory power of these EcoRV variants through the choice of appropriate reaction conditions, in particular low salt concentration and low reaction temperatures. It must be emphasized that the enhanced selectivity of these EcoRV variants toward EcoRV sites in a flexible and nonflexible sequence context, respectively, is not only seen with oligodeoxynucleotides, but also with plasmid substrates.

Base Sequence↗
PubMed1998

Kinetic characterization of linear diffusion of the restriction endonuclease EcoRV on DNA.

We have examined the kinetic parameters of linear diffusion of EcoRV on DNA. The data were analyzed by Monte Carlo simulations in which the efficiency of recognition of EcoRV sites during linear diffusion, the efficiency of linear diffusion, and the behavior of enzymes at the ends of linear DNA is explicitly treated. The analysis of the dependence of linear diffusion on the concentrations of NaCl and MgCl2 shows that linear diffusion is maximal at 50 mM NaCl under all concentrations of MgCl2 tested and increases with increasing concentrations of Mg2+ up to 10 mM, the highest concentration used in the test. Under these conditions, EcoRV scans 2 x 10(6) bp during one binding event with a velocity of about 1.7 x 10(6) bp s-1. The enzyme tends to overlook cleavage sites at 1 mM but not at 10 mM MgCl2. This result confirms the thermodynamic finding that EcoRV does not bind very specifically to DNA in the absence of Mg2+. It demonstrates that there is a Mg2+-dependent continuous transition between a nonspecific and a specific binding mode of EcoRV to DNA. By comparing cleavage rates of linear DNA whose ends are free or blocked, we have shown that EcoRV has a very low probability to fall off at the ends of linear DNA. The enzyme rather is "reflected" and continues linear diffusion. EcoRV does not cleave oligonucleotides containing two EcoRV sites processively. Consequently, dissociation of the enzyme from the cleavage products is not preceded by a transfer to nonspecific DNA, and linear diffusion is not involved in product dissociation in EcoRV.

Base Sequence↗
PubMed1998

Biochemical characterization of Anabaena sp. strain PCC 7120 non-specific nuclease NucA and its inhibitor NuiA.

We have established overexpression systems and purification protocols for NucA and NuiA, a sugar non-specific nuclease and its protein inhibitor from Anabaena sp. strain PCC 7120, in order to characterize these proteins in detail. CD spectroscopy revealed that NucA has a similar secondary-structure composition, 13% alpha helix and 20% beta sheet, to the related Serratia nuclease, while NuiA represents a protein with a higher alpha-helical (29%) and beta-sheet (24%) content than NucA. Denaturation experiments showed that the stabilities of NucA and NuiA are in the typical range for proteins of mesophilic organisms, NuiA with deltaG0H2O = 63.4 J x mol(-1)residue, being slightly more stable than its target NucA with delta deltaG0H2O = 46.3 J x mol(-1)residue. The nuclease requires divalent metal ions as cofactors, the optimum concentration being around 5 mM for Mn2+ or Mg2+. The order of effectiveness of various divalent cations to function as cofactors for the hydrolytic activity of NucA is Mn2+ = Co2+ > Mg2+ > or = Ni2+ >> or Ca2+ = Cd2+ at a concentration of 5 mM. Nuclease activity decreases with increasing concentration of monovalent salt. The activity of NucA shows a pH optimum at pH 5.5-7.5. The temperature optimum is around 35 degrees C, the activation energy was calculated to be 53 kJ mol(-1). The specific activity of the nuclease towards high molecular-mass DNA is 8.4 x 10(6) Kunitz-units x mg(-1), which means that NucA is one of the most active nucleases known. Kinetic constants for the cleavage of various DNA and RNA substrates by NucA are all in the range Km < or = 0.1 mg x ml(-1) and k(cat) approximately 1000 s(-1). As other non-specific nucleases, NucA exhibits sequence preferences, similar to the related Serratia nuclease, NucA avoids cleavage of d(A) x d(T) tracts. The nucleolytic activity of NucA is completely inhibited at equimolar concentrations of nuclease and inhibitor. An ultracentrifugation analysis showed that NucA and NuiA form a 1:1 complex. The interaction of NucA with NuiA was also investigated by CD spectroscopy and revealed no major conformational changes upon complex formation of the two proteins.

Amino Acid Sequence↗
PubMed1998

Towards the design of rare cutting restriction endonucleases: using directed evolution to generate variants of EcoRV differing in their substrate specificity by two orders of magnitude.

The restriction endonuclease EcoRV cleaves DNA highly specifically within GATATC sequences. In order to create EcoRV variants that have an extended recognition site we have employed a semi-rational random mutagenesis/selection procedure. Twenty-two amino acid residues were subjected to random mutagenesis and about 500 EcoRV variants representing three generations of mutants were screened. Among these some highly active variants that strongly prefer AT-flanked cleavage sites (e.g. S183A/Q224R, T93S/I103F/S183A/T222S or N97T/S183A/T222S) and others that prefer GC flanks (e.g. K104N/A181T) were identified. As wild-type EcoRV does not discriminate between these cleavage sites, the generation of these variants represents a significant first step towards redesigning EcoRV to become an 8 or 10 bp cutter. Such enzymes, only very rarely found in nature, could be extremely helpful for the manipulation of large DNA fragments.

Deoxyribonucleases, Type II Site-Specific↗
PubMed1997

Does the restriction endonuclease EcoRV employ a two-metal-Ion mechanism for DNA cleavage?

Two models for the catalytic mechanism of the restriction endonuclease EcoRV exist which differ in the number and function of metal ions proposed to be directly involved in catalysis. In one model, two metal ions bound by Glu45, Asp74, and Asp90 are assumed to have a direct catalytic function; in the other, only one metal ion bound by Asp74 and Asp90. We show here that in the presence of Mn2+, the catalytic activity of an EcoRV-E45A mutant is only slightly reduced (1.8-fold) as compared to wild type EcoRV and that the single-turnover rate constant of DNA cleavage by E45A is reduced only 39-fold, whereas the D74A and D90A mutants are catalytically inactive under all conditions. These findings make an important catalytic function of Glu45, like binding of an essential divalent metal ion, unlikely. In addition, we have analyzed the dependence of the DNA cleavage rate by EcoRV and EcoRV mutants on the concentration of Mg2+ and Mn2+. We found for the wild type enzyme a sigmoidal dependence of the rate of DNA cleavage on the concentration of Mg2+ or Mn2+, indicative of at least two metal ions involved in DNA binding and catalysis. This, however, does not mean that EcoRV follows a two-metal-ion mechanism in DNA cleavage, because also for the E45A mutant a sigmoidal dependence of the rate of DNA cleavage on the Mg2+ concentration was found, making metal ion binding to the E45/D74 site unlikely. In contrast, the Y219C mutant shows a hyperbolic dependence. In agreement with results obtained earlier, these findings demonstrate binding of a Mg2+ ion at a site influenced by Tyr219, an amino acid residue that is far away from the active site. Metal binding at this site does not have a catalytic role but rather supports specific DNA binding. We conclude that on the basis of our data a two-metal-ion mechanism of DNA cleavage is unlikely for EcoRV and that the complex metal ion effects observed are due to metal ion binding at sites that are not directly involved in catalysis.

Bacteriophage lambda↗
PubMed1997

Characterization of the interaction between the restriction endonuclease McrBC from E. coli and its cofactor GTP.

McrBC, a GTP-dependent restriction enzyme from E. coli K-12, cleaves DNA containing methylated cytosine residues 40 to 80 residues apart and 3'-adjacent to a purine residue (PumCN40-80PumC). The presence of the three consensus sequences characteristic for guanine nucleotide binding proteins in one of the two subunits of McrBC suggests that this subunit is responsible for GTP binding and hydrolysis. We show here that (i) McrB binds GTP with an affinity of 10(6) M-1 and that GTP binding stabilizes McrB against thermal denaturation. (ii) McrB binds GDP about 50-fold and ATP at least three orders of magnitude more weakly than GTP. (iii) McrB hydrolyzes GTP in the presence of Mg2+ with a steady-state rate of approximately 0.5 min-1. (iv) McrC stimulates GTP hydrolysis 30-fold, but substrate DNA has no detectable effect on the GTPase activity of McrB, neither by itself nor in the presence of McrC. (v) Substitution of N339 and N376 with alanine allowed us to identify NTAD (339 to 342) rather than NKKA (376 to 379) as the equivalent of the third consensus sequence motif characteristic for guanine nucleotide binding proteins, NKXD.

Adenosine Triphosphate↗
PubMed1997

Recognition and cleavage of DNA by type-II restriction endonucleases.

Restriction endonucleases are enzymes which recognize short DNA sequences and cleave the DNA in both strands. Depending on the enzymological properties different types are distinguished. Type II restriction endonucleases are homodimers which recognize short palindromic sequences 4-8 bp in length and, in the presence of Mg2+, cleave the DNA within or next to the recognition site. They are capable of non-specific binding to DNA and make use of linear diffusion to locate their target site. Binding and recognition of the specific site involves contacts to the bases of the recognition sequence and the phosphodiester backbone over approximately 10-12 bp. In general, recognition is highly redundant which explains the extreme specificity of these enzymes. Specific binding is accompanied by conformational changes over both the protein and the DNA. This mutual induced fit leads to the activation of the catalytic centers. The precise mechanism of cleavage has not yet been established for any restriction endonuclease. Currently two models are discussed: the substrate-assisted catalysis mechanism and the two-metal-ion mechanism. Structural similarities identified between EcoRI, EcoRV, BamHI, PvuII and Cfr10I suggest that many type II restriction endonucleases are not only functionally but also evolutionarily related.

Binding Sites↗
PubMed1997

The recognition of methylated DNA by the GTP-dependent restriction endonuclease McrBC resides in the N-terminal domain of McrB.

McrBC is a GTP-dependent restriction endonuclease of E. coli K12, selectively directed against DNA containing modified cytosine residues. McrB, one of its components, is responsible for the binding and, together with McrC, for the cleavage of DNAs containing two 5'-Pu(m)C sites separated by 40-80 base pairs. Gel retardation assays with wild-type and mutant McrB reveal that (i) single 5'-Pu(m)C sites in DNA can be sufficient to elicite binding by McrB. Binding to such substrates is, however, weak and strongly dependent on the sequence context of Pu(m)C sites. (ii) Strong DNA binding (K(ass) approximately 10(7)M[-1]) is dependent on the presence of at least two Pu(m)C sites, even if they are separated by less than 40 bp, and is modulated by the sequence context (-A(m)CCGGT- --> -A(m)CT(C/G)AGT- --> -AGG(m)CCT- --> -AAG(m)CTT-). (iii) DNA binding by McrB is accompanied by formation of distinct multiple complexes whose distribution is modulated by GTP. (iv) McrC, which cannot bind DNA by itself, moderately stimulates the DNA binding of McrB and converts McrB-DNA complexes to large aggregates. (v) Deletion of the C-terminal half of McrB, which harbors the three consensus sequences characteristic for guanine nucleotide binding proteins, leads to protein inactive in GTP binding and/or hydrolysis and in McrC-assisted DNA cleavage; the protein, however, remains fully competent in DNA binding. (vi) Mutations in McrB which lead to a reduction in GTP binding and/or hydrolysis can affect DNA binding, suggesting that the two activities are coupled in the full-length protein.

Base Composition↗