Mechanistic Insight into the Catalytic Activity of ββα‐Metallonucleases from Computer Simulations: <i>Vibrio vulnificus</i> Periplasmic Nuclease as a Test Case

Bueren-Calabuig, Juan A.; Coderch, Claire; Rico, Eva; Jiménez-Ruı́z, Antonio; Gago, Federico

doi:10.1002/cbic.201100485

Cited by 11 publications

(20 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this regard, recent progress in the implementation of hybrid QM/MM methods [32][33][34][35] has allowed us to address several biologically relevant questions concerning reactivity in macromolecules by taking into account the full protein environment during a chemical reaction. [36][37][38][39] The present results strongly support the view that the protein environment in tetrameric HAL is crucial for exerting the required compression on the reactive Ala142-Ser143-Gly144 loop and bringing into close proximity residues 142 and 144 in a suitable orientation ( Figure 1A). MD simulations showed that the near-attack conformation for this loop is achieved only when the full tetramer in explicit water is employed and not when a single HAL monomer or a HAL dimer are considered.…”

Section: Discussionsupporting

confidence: 85%

“…24 Firstly, solvent molecules and counterions were relaxed by energy minimization and allowed to redistribute around the positionally restrained solute atoms (25 Kcal mol -1 Å -2 ) at constant temperature and pressure (1 atm), essentially as described previously. 25 These harmonic restraints were gradually reduced until they were completely removed. The resulting system was then heated from 100 to 300 K during 20 ps, equilibrated at 300 K for 100 ps and further simulated under the same conditions up to a total time of 60 ns, during which system coordinates were collected every 20 ps for further analysis.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Stepwise Simulation of 3,5-Dihydro-5-methylidene-4H-imidazol-4-one (MIO) Biogenesis in Histidine Ammonia-lyase

et al. 2016

Self Cite

View full text Add to dashboard Cite

A 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) electrophilic moiety is post-translationally and autocatalytically generated in homotetrameric histidine ammonia-lyase (HAL) and other enzymes containing the tripeptide Ala-Ser-Gly in a suitably positioned loop. The backbone cyclization step is identical to that taking place during fluorophore formation in green fluorescent protein from the tripeptide Ser-Tyr-Gly, but dehydration, rather than dehydrogenation by molecular oxygen, is the reaction that gives rise to the mature MIO ring system. To gain additional insight into this unique process and shed light on some still unresolved issues, we have made use of extensive molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations implementing the self-consistent charge density functional tight-binding method on a fully solvated tetramer of Pseudomonas putida HAL. Our results strongly support the idea that mechanical compression of the reacting loop by neighboring protein residues in the precursor state is absolutely required to prevent formation of inhibitory main-chain hydrogen bonds and to enforce proper alignment of donor and acceptor orbitals for bond creation. The consideration of the protein environment in our computations shows that water molecules, which have been mostly neglected in previous theoretical work, play a highly relevant role in the reaction mechanism and, more importantly, that backbone cyclization resulting from the nucleophilic attack of the Gly amide lone pair on the π* orbital of the Ala carbonyl precedes side-chain dehydration of the central serine.

show abstract

Section: Discussionsupporting

confidence: 85%

Section: Methodsmentioning

confidence: 99%

Stepwise Simulation of 3,5-Dihydro-5-methylidene-4H-imidazol-4-one (MIO) Biogenesis in Histidine Ammonia-lyase

et al. 2016

Self Cite

View full text Add to dashboard Cite

show abstract

“…This would be in agreement with the recently proposed shuttle mechanism according to which the leaving group is protonated by the hydrogen ion originating from the same water molecule that initiated the nucleophilic attack. 61 The above mechanism would be similar to the one found in a serine recombinase-mediated DNA cleavage. 62 Although arginine is rarely mentioned in the literature to behave as an acid but the environment of an enzyme active site can significantly shift the pK a values of critical residues.…”

Section: Discussionmentioning

confidence: 93%

A new insight into the zinc-dependent DNA-cleavage by the colicin E7 nuclease: a crystallographic and computational study

et al. 2014

View full text Add to dashboard Cite

The nuclease domain of colicin E7 metallonuclease (NColE7) contains its active centre at the C-terminus. The mutant ΔN4-NColE7-C* - where the four N-terminal residues including the positively charged K446, R447 and K449 are replaced with eight residues from the GST tag - is catalytically inactive. The crystal structure of this mutant demonstrates that its overall fold is very similar to that of the native NColE7 structure. This implicates the stabilizing effect of the remaining N-terminal sequence on the structure of the C-terminal catalytic site and the essential role of the deleted residues in the mechanism of the catalyzed reaction. Complementary QM/MM calculations on the protein-DNA complexes support the less favourable cleavage by the mutant protein than by NColE7. Furthermore, a water molecule as a possible ligand for the Zn(2+)-ion is proposed to play a role in the catalytic process. These results suggest that the mechanism of the Zn(2+)-containing HNH nucleases needs to be further studied and discussed.

show abstract

“…As the presence of this residue or any positively charged residue potentially replacing proved to be essential for the catalytic activity of the HNH motif [129,130,132] it can also be considered to participate directly in the catalytic mechanism. Being a flexible residue, it may facilitate the proton transfer from the histidine general base towards the leaving alcoholate [133]. Such a role has been suggested recently in other hydrolytic enzymes [134,135].…”

Section: Nuclease Colicinsmentioning

confidence: 94%

Rescue of the activity of HNH nuclease mutants - towards controlled enzymes for gene therapy

Gyurcsik

2016

CPPS

View full text Add to dashboard Cite

Artificial nucleases are designed for in vivo gene engineering, as the DNA cleavage performed at a specific target site enhances the effectiveness of the cell's DNA repair machinery. The therapeutic potential of the above phenomenon stems from the knowledge that (i) the shifted reading frame can be restored by non-homologous end-joining, or (ii) a DNA of erroneous sequence -causing a genetic disease -can be corrected by homologous recombination in the presence of a suitable DNA template. Besides the advantageous properties of the nowadays applied zinc finger nucleases, TALE nucleases and the CRISPR/Cas9 system, they possess a residual citotoxicity. This is related to offtarget cleavages, which could be prevented by the strict regulation of the enzymes. The studies on enzymes acting naturally in a controlled manner are beneficial to get better insight into their mechanism. Such enzymes or their appropriate domains may be the most promising alternatives to the presently applied ones. As an example, the DNA cleavage of the inactive HNH nuclease mutants is inducible in a multiple way. This property may be used for establishing a control mechanism and thus, in combination with specific DNA-binding domains they are good candidates for the catalytic site of artificial nucleases. Here we collect the results on the properties of the HNH nucleases that allow for their redesign into enzymes with possible therapeutic applications.

show abstract

Mechanistic Insight into the Catalytic Activity of ββα‐Metallonucleases from Computer Simulations: Vibrio vulnificus Periplasmic Nuclease as a Test Case

Cited by 11 publications

References 43 publications

Stepwise Simulation of 3,5-Dihydro-5-methylidene-4H-imidazol-4-one (MIO) Biogenesis in Histidine Ammonia-lyase

Stepwise Simulation of 3,5-Dihydro-5-methylidene-4H-imidazol-4-one (MIO) Biogenesis in Histidine Ammonia-lyase

A new insight into the zinc-dependent DNA-cleavage by the colicin E7 nuclease: a crystallographic and computational study

Rescue of the activity of HNH nuclease mutants - towards controlled enzymes for gene therapy

Contact Info

Product

Resources

About