Role of magnesium ions in DNA recognition by the EcoRV restriction endonuclease

Zahran, Mai; Bereźniak, Tomasz; Imhof, Petra; Smith, Jeremy C.

doi:10.1016/j.febslet.2011.07.036

Cited by 13 publications

(24 citation statements)

References 31 publications

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“…Literature reports that Mg 2+ plays a functional role in the catalytic mechanism and the stability of protein-DNA complex. Metal ions also lower the local pKa, and this, considering the harsh environment that the organism experiences, is in a good agreement with our study [ 7 , 8 , 41 – 43 ].…”

Section: Resultssupporting

confidence: 92%

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Role of magnesium ions in the reaction mechanism at the interface between Tm1631 protein and its DNA ligand

Ogrizek

Konc

Bren

et al. 2016

Chemistry Central Journal

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A protein, Tm1631 from the hyperthermophilic organism Thermotoga maritima belongs to a domain of unknown function protein family. It was predicted that Tm1631 binds with the DNA and that the Tm1631–DNA complex is an endonuclease repair system with a DNA repair function (Konc et al. PLoS Comput Biol 9(11): e1003341, 2013). We observed that the severely bent, strained DNA binds to the protein for the entire 90 ns of classical molecular dynamics (MD) performed; we could observe no significant changes in the most distorted region of the DNA, where the cleavage of phosphodiester bond occurs. In this article, we modeled the reaction mechanism at the interface between Tm1631 and its proposed ligand, the DNA molecule, focusing on cleavage of the phosphodiester bond. After addition of two Mg2+ ions to the reaction center and extension of classical MD by 50 ns (totaling 140 ns), the DNA ligand stayed bolted to the protein. Results from density functional theory quantum mechanics/molecular mechanics (QM/MM) calculations suggest that the reaction is analogous to known endonuclease mechanisms: an enzyme reaction mechanism with two Mg2+ ions in the reaction center and a pentacovalent intermediate. The minimum energy pathway profile shows that the phosphodiester bond cleavage step of the reaction is kinetically controlled and not thermodynamically because of a lack of any energy barrier above the accuracy of the energy profile calculation. The role of ions is shown by comparing the results with the reaction mechanisms in the absence of the Mg2+ ions where there is a significantly higher reaction barrier than in the presence of the Mg2+ ions.Graphical abstractA protein, Tm1631 from the hyperthermophilic organism Thermotoga maritima belongs to a domain of unknown function protein family. We modeled the reaction mechanism at the interface between Tm1631 and its proposed ligand, the DNA molecule, focusing on cleavage of the phosphodiester bondElectronic supplementary materialThe online version of this article (doi:10.1186/s13065-016-0188-6) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 92%

“…Endonucleases require one, two or three divalent metal ions, such as Mn 2+ or Mg 2+ in the catalytic site [ 7 – 13 ]. It is believed that in thermal environment, such as the one Thermotoga maritima lives in at temperatures around 80 °C, the most suitable metal ion for this kind of system is Mg 2+ [ 14 ].…”

Section: Introductionmentioning

confidence: 99%

Role of magnesium ions in the reaction mechanism at the interface between Tm1631 protein and its DNA ligand

Ogrizek

Konc

Bren

et al. 2016

Chemistry Central Journal

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“…[29] Theoretical study by Zahran et al shows that the second magnesium ion serves to stabilize the distorted DNA. [14] Our observations on the stability of the aspartic acids is qualitatively consistent to this theoretical report. Earlier studies show [38][39][40][41] that the residues which get conformationally destabilized and disordered participate in ligand binding.…”

Section: The Conformational Free Energy ðδGsupporting

confidence: 91%

“…performed to understand role of Mg 2+ as natural cofactor. [11,[14][15][16][17] But the microscopic origin of the specificity of metal ion cofactor Mg 2 + vis-à-vis Ca 2+ in DNA cleavage is unknown. [18][19][20] Naturally occurring restriction endonucleases are classified as four types such as type I, II, III and IV, depending on their structure, recognition site, cleavage site etc.…”

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confidence: 99%

Microscopic insight to specificity of metal ion cofactor in DNA cleavage by restriction endonuclease EcoRV

et al. 2020

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Restriction endonucleases protect bacterial cells against bacteriophage infection by cleaving the incoming foreign DNA into fragments. In presence of Mg 2+ ions, EcoRV is able to cleave the DNA but not in presence of Ca 2+ , although the protein binds to DNA in presence of both metal ions. We make an attempt to understand this difference using conformational thermodynamics. We calculate the changes in conformational free energy and entropy of conformational degrees of freedom, like DNA base pair steps and dihedral angles of protein residues in Mg 2+ (A)-EcoRV-DNA complex compared to Ca 2+ (S)-EcoRV-DNA complex using all-atom molecular dynamics (MD) trajectories of the complexes. We find that despite conformational stability and order in both complexes, the individual degrees of freedom behave differently in the presence of two different metal ions. The base pairs in cleavage region are highly disordered in Ca 2+ (S)-EcoRV-DNA compared to Mg 2+ (A)-EcoRV-DNA. One of the acidic residues ASP90, coordinating to the metal ion in the vicinity of the cleavage site, is conformationally destabilized and disordered, while basic residue LYS92 gets conformational stability and order in Ca 2+ (S) bound complex than in Mg 2+ (A) bound complex. The enhanced fluctuations hinder placement of the metal ion in the vicinity of the scissile phosphate of DNA. Similar loss of conformational stability and order in the cleavage region is observed by the replacement of the metal ion. Considering the placement of the metal ion near scissile phosphate as requirement for cleavage action, our results suggest that the changes in conformational stability and order of the base pair steps and the protein residues lead to cofactor sensitivity of the enzyme. Our method based on fluctuations of microscopic conformational variables can be applied to understand enzyme activities in other protein-DNA systems.

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“…Available high-resolution X-ray structures of many endonuclease enzymes in complex with target DNA or RNA or substrate analogues [31][32][33][34][35] and various biochemical studies have shown that metal cofactors are essential for the enzymatic phosphodiester cleavage [36][37][38][39]. The metal ions not only play a direct role in the chemical step but also influence the binding interaction of the enzyme with the DNA or RNA [40,41]. For some endonucleases different structures with different numbers of metal ions have been solved by X-ray crystallography under varying conditions [42][43][44][45][46][47].…”

Section: Introductionmentioning

confidence: 99%

Phosphodiester hydrolysis computed for cluster models of enzymatic active sites

Batebi

Imhof

2016

Theor Chem Acc

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Computation of phosphodiester hydrolysis in different models with one or two metal ions, representing typical active site architectures of nucleases, reveal an associative mechanism to be favorable in all of the cases studied in this work. Direct attack of the nucleophilic water molecule with proton transfer to the phosphate group is facilitated by an extra positive charge as provided by a metal ion located at the attack site or a positively charged histidine residue whereas no such contribution can be observed on leaving group departure. A major catalytic effect is found by proton transfer from the nucleophilic water molecule to a histidine-aspartate cluster. Attack of the thus generated hydroxide ion on the phosphate group is just sufficiently stabilized by the metal ions to allow subsequent P-O bond dissociation.

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Role of magnesium ions in DNA recognition by the EcoRV restriction endonuclease

Cited by 13 publications

References 31 publications

Role of magnesium ions in the reaction mechanism at the interface between Tm1631 protein and its DNA ligand

Role of magnesium ions in the reaction mechanism at the interface between Tm1631 protein and its DNA ligand

Microscopic insight to specificity of metal ion cofactor in DNA cleavage by restriction endonuclease EcoRV

Phosphodiester hydrolysis computed for cluster models of enzymatic active sites

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