0000000000210619
AUTHOR
Juan Aranda
The Catalytic Mechanism of Carboxylesterases: A Computational Study
The catalytic mechanism of carboxylesterases (CEs, EC 3.1.1.1) is explored by computational means. CEs hydrolyze ester, amide, and carbamate bonds found in xenobiotics and endobiotics. They can also perform transesterification, a reaction important, for instance, in cholesterol homeostasis. The catalytic mechanisms with three different substrates (ester, thioester, and amide) have been established at the M06-2X/6-311++G**//B3LYP/6-31G* level of theory. It was found that the reactions proceed through a mechanism involving four steps instead of two as is generally proposed: (i) nucleophilic attack of serine to the substrate, forming the first tetrahedral intermediate, (ii) formation of the ac…
Regioselectivity of the OH Radical Addition to Uracil in Nucleic Acids. A Theoretical Approach Based on QM/MM Simulations.
Oxidation of nucleic acids is ubiquitous in living beings under metabolic impairments and/or exposed to external agents such as radiation, pollutants, or drugs, playing a central role in the development of many diseases mediated by DNA/RNA degeneration. Great efforts have been devoted to unveil the molecular mechanisms behind the OH radical additions to the double bonds of nucleobases; however, the specific role of the biological environment remains relatively unexplored. The present contribution tackles the study of the OH radical addition to uracil from the gas phase to a full RNA macromolecule by means of quantum-chemistry methods combined with molecular dynamics simulations. It is shown…
Substrate promiscuity in DNA methyltransferase M.PvuII. A mechanistic insight
M.PvuII is a DNA methyltransferase from the bacterium Proteus vulgaris that catalyzes methylation of cytosine at the N4 position. This enzyme also displays promiscuous activity catalyzing methylation of adenine at the N6 position. In this work we use QM/MM methods to investigate the reaction mechanism of this promiscuous activity. We found that N6 methylation in M.PvuII takes place by means of a stepwise mechanism in which deprotonation of the exocyclic amino group is followed by the methyl transfer. Deprotonation involves two residues of the active site, Ser53 and Asp96, while methylation takes place directly from the AdoMet cofactor to the target nitrogen atom. The same reaction mechanism…
Singlet Oxygen Attack on Guanine: Reactivity and Structural Signature within the B-DNA Helix
International audience; Oxidatively generated DNA lesions are numerous and versatile, and have been the subject of intensive research since the discovery of 8-oxoguanine in 1984. Even for this prototypical lesion, the precise mechanism of formation remains elusive due to the inherent difficulties in characterizing high-energy intermediates. We have probed the stability of the guanine endoperoxide in B-DNA as a key intermediate and determined a unique activation free energy of around 6 kcal mol−1 for the formation of the first C−O covalent bond upon the attack of singlet molecular oxygen (1O2) on the central guanine of a solvated 13 base-pair poly(dG-dC), described by means of quantum mechan…
Free energy profiles for two ubiquitous damaging agents: methylation and hydroxylation of guanine in B-DNA
International audience; DNA methylation and hydroxylation are two ubiquitous reactions in DNA damage induction, yet insights are scarce concerning the free energy of activation within B-DNA. We resort to multiscale simulations to investigate the attack of a hydroxyl radical and of the primary diazonium onto a guanine embedded in a solvated dodecamer. Reaction free energy profiles characterize two strongly exergonic processes, yet allow unprecedented quantification of the barrier towards this damage reaction, not higher than 6 kcal mol−1 and sometimes inexistent, and of the exergonicities. In the case of the [G(C8)-OH]˙ intermediate, we challenge the functional dependence of such simulations…
Mechanism of sulfur transfer across protein-protein interfaces: The cysteine desulfurase model system
CsdA cysteine desulfurase (the sulfur donor) and the CsdE sulfur acceptor are involved in biological sulfur trafficking and in iron-sulfur cluster assembly in the model bacterium Escherichia coli. CsdA and CsdE form a stable complex through a polar interface that includes CsdA Cys328 and CsdE Cys61, the two residues known to be involved in the sulfur transfer reaction. Although mechanisms for the transfer of a sulfur moiety across protein-protein interfaces have been proposed based on the IscS-IscU and IscS-TusA structures, the flexibility of the catalytic cysteine loops involved has precluded a high resolution view of the active-site geometry and chemical environment for sulfur transfer. H…
Molecular Mechanism of Inhibition of DNA Methylation by Zebularine
In this work, we have analyzed the molecular mechanism of inhibition of a C5-DNA methyltransferase by zebularine using classical and QM/MM simulations. We found that the reaction proceeds with the addition of an unprotonated cysteine to the C6 position of the ring followed by methyl transfer to the C5 position. However, while the first step is reversible and presents a moderate free-energy barrier, the second step presents a large free-energy barrier, preventing the formation of the methylated complex. This mechanistic proposal agrees with recent experimental observations that point to the formation of a reversible covalent complex between DNA containing zebularine and methyltransferases. T…
Modeling methods for studying post-translational and transcriptional modifying enzymes.
Biological catalysis is a complex chemical process that involves not only electronic reorganization in the substrate but also the reorganization of the catalyst. This complexity is even larger in the case of post-transcriptional and post-translational modifications which may involve the interaction between two biomacromolecules. However, the development over the past decades of new computational methods and strategies is offering a detailed molecular picture of the catalytic event and a deep understanding of the mechanisms of chemical reactions in biological environments. Here we review the efforts made in the last years to model catalysis in post-transcriptional and post-translational proc…
Unraveling the Reaction Mechanism of Enzymatic C5-Cytosine Methylation of DNA. A Combined Molecular Dynamics and QM/MM Study of Wild Type and Gln119 Variant
M.HhaI is a DNA methyltransferase from Haemophilus hemolyticus that catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to the C5 position of a cytosine. This enzyme is a paradigmatic model for C5 DNA methyltransferases due to its major homology to mammalian enzymes and to the availability of high-resolution structures of the DNA–enzyme complex. In spite of the number of experimental and theoretical analyses carried out for this system, many mechanistic details remain unraveled. We have used full atomistic classical molecular dynamics simulations to explore the protein–SAM–DNA ternary complex, where the target cytosine base is flipped out into the active site for bot…
Dynamics and reactivity in Thermus aquaticus N6-adenine methyltransferase.
M.TaqI is a DNA methyltransferase from Thermus aquaticus that catalyzes the transfer of a methyl group from S-adenosyl-l-methionine to the N6 position of an adenine, a process described only in prokaryotes. We have used full atomistic classical molecular dynamics simulations to explore the protein–SAM–DNA ternary complex where the target adenine is flipped out into the active site. Key protein–DNA interactions established by the target adenine in the active site are described in detail. The relaxed structure was used for a combined quantum mechanics/molecular mechanics exploration of the reaction mechanism using the string method. According to our free energy calculations the reaction takes…
Insights into the inhibited form of the redox-sensitive SufE-like sulfur acceptor CsdE
17 p.-8 fig.
Theoretical Study of the Catalytic Mechanism of DNA-(N4-Cytosine)-Methyltransferase from the Bacterium Proteus vulgaris
In this paper the reaction mechanism for methylation of cytosine at the exocyclic N4 position catalyzed by M.PvuII has been explored by means of hybrid quantum mechanics/molecular mechanics (QM/MM) methods. A reaction model was prepared by placing a single cytosine base in the active site of the enzyme. In this model the exocyclic amino group of the base establishes hydrogen bond interactions with the hydroxyl oxygen atom of Ser53 and the carbonyl oxygen atom of Pro54. The reaction mechanism involves a direct methyl transfer from AdoMet to the N4 atom and a proton transfer from this atom to Ser53, which in turn transfers a proton to Asp96. Different timings for the proton transfers and meth…