Ruibo Wu

A Proton-Shuttle Reaction Mechanism for Histone Deacetylase 8 and the Catalytic Role of Metal Ions

Zinc-dependent histone deacetylase 8 (HDAC8) catalyzes the removal of acetyl moieties from histone tails, and is critically involved in regulating chromatin structure and gene expression. The detailed knowledge of its catalytic process is of high importance since it has been established as a most promising target for the development of new antitumor drugs. By employing Born-Oppenheimer ab initio QM/MM molecular dynamics simulations and umbrella sampling, a state-of-the-art approach to simulate enzyme reactions, we have provided further evidence against the originally proposed general acid-base catalytic pair mechanism for Zinc-dependent histone deacetylases. Instead, our results indicated that HDAC8 employs a proton-shuttle catalytic mechanism, in which a neutral His143 first serves as the general base to accept a proton from the zinc-bound water molecule in the initial rate-determining nucleophilic attack step, and then shuttles it to the amide nitrogen atom to facilitate the cleavage of the amide bond. During the deacetylation process, the Zn(2+) ion changes its coordination mode and plays multiple catalytic roles. For the K(+) ion, which is located about 7 A from the catalytic Zn(2+) ion and conserved in class I and II HDACs, our simulations indicated that its removal would lead to the different transition state structure and a higher free energy reaction barrier for the rate-determining step. It is found that the existence of this conserved K(+) ion would enhance the substrate binding, increase the basicity of His143, strengthen the catalytic role of zinc ion, and improve the transition state stabilization by the enzyme environment.

Zinc Chelation with Hydroxamate in Histone Deacetylases Modulated by Water Access to the Linker Binding Channel

It is of significant biological interest and medical importance to develop class- and isoform-selective histone deacetylase (HDAC) modulators. The impact of the linker component on HDAC inhibition specificity has been revealed but is not understood. Using Born-Oppenheimer ab initio QM/MM MD simulations, a state-of-the-art approach to simulating metallo-enzymes, we have found that the hydroxamic acid remains to be protonated upon its binding to HDAC8, and thus disproved the mechanistic hypothesis that the distinct zinc-hydroxamate chelation modes between two HDAC subclasses come from different protonation states of the hydroxamic acid. Instead, our simulations suggest a novel mechanism in which the chelation mode of hydroxamate with the zinc ion in HDACs is modulated by water access to the linker binding channel. This new insight into the interplay between the linker binding and the zinc chelation emphasizes its importance and gives guidance regarding linker design for the development of new class-IIa-specific HDAC inhibitors.

Computational Design of a Time-Dependent Histone Deacetylase 2 Selective Inhibitor

Development of isoform-selective histone deacetylase (HDAC) inhibitors is of great biological and medical interest. Among 11 zinc-dependent HDAC isoforms, it is particularly challenging to achieve isoform inhibition selectivity between HDAC1 and HDAC2 due to their very high structural similarities. In this work, by developing and applying a novel de novo reaction-mechanism-based inhibitor design strategy to exploit the reactivity difference, we have discovered the first HDAC2-selective inhibitor, β-hydroxymethyl chalcone. Our bioassay experiments show that this new compound has a unique time-dependent selective inhibition on HDAC2, leading to about 20-fold isoform-selectivity against HDAC1. Furthermore, our ab initio QM/MM molecular dynamics simulations, a state-of-the-art approach to study reactions in biological systems, have elucidated how the β-hydroxymethyl chalcone can achieve the distinct time-dependent inhibition toward HDAC2.

Combined quantum mechanics/molecular mechanics study on the reversible isomerization of glucose and fructose catalyzed by Pyrococcus furiosus phosphoglucose isomerase

Phosphoglucose isomerase (PGI), which catalyzes the reversible interconversion of glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P), is represented by two evolutionarily distinct protein families. One is a conventional type in eubacteria, eukaryotes, and a few archaea, where the active sites contain no metal ions and reactions proceed via the cis-enediol intermediate mechanism. The second type, found recently in euryarchaeota species, belongs to metalloenzymes, and controversies exist over whether the catalyzed isomerization occurs via the cis-enediol intermediate mechanism or a hydride shift mechanism. We studied the reversible interconversion of the open-chain form G6P and F6P catalyzed by the metal-containing Pyrococcus furiosus PGI by performing QM(B3LYP)/MM single-point optimizations and QM(PM3)/MM molecular dynamics simulations. A zwitterion intermediate-based mechanism, which involves both proton and hydride transfers, has been put forward. The presence of the key zwitterionic intermediate in this mechanism can effectively reconcile the controversial mechanisms and rationalize the enzymatic reaction. Computations show that the overall isomerization process is quite facile, both dynamically and thermodynamically. The crucial roles of conserved residues have been elucidated on the basis of computations on their alanine mutants. In particular, Tyr152 pushes the H1 transfer through a hydride-shift mechanism and dominates the stereochemistry selectivity of the hydrogen transfer. The rest of the conserved residues basically maintain the substrate in the near-attack reactive conformation and mediate the proton transfer. Although Zn(2+) is not directly involved in the reaction, the metal ion as a structural anchor constructs a hydrogen bond wire to connect the substrate to the outer region, providing a potential channel for hydrogen exchange between the substrate and solvent.

QM/MM Molecular Dynamics Study of Purine-Specific Nucleoside Hydrolase

Although various T. vivax purine-specific inosine-adenosine-guanosine nucleoside hydrolase (IAG-NH) crystal structures were determined in recent years, the mechanistic details for the cleavage of N-glycosidic bond and the release of base are still unclear. Herein, the irreversible hydrolysis reaction has been studied by ab initio QM/MM MD simulations, and the results indicate a highly dissociative and concerted mechanism. The protonation of substrate at N7 of inosine is found to strongly facilitate the hydrolysis process, while the hydrolysis reaction is less sensitive to the protonation state of Asp 40 residue. The proton-transfer channel and the dependence of activity on the anti/syn-conformation of substrate are also explored.

Molecular dynamics simulations of the detoxification of paraoxon catalyzed by phosphotriesterase.

Combined QM(PM3)/MM molecular dynamics simulations together with QM(DFT)/MM optimizations for key configurations have been performed to elucidate the enzymatic catalysis mechanism on the detoxification of paraoxon by phosphotriesterase (PTE). In the simulations, the PM3 parameters for the phosphorous atom were reoptimized. The equilibrated configuration of the enzyme/substrate complex showed that paraoxon can strongly bind to the more solvent‐exposed metal ion Znβ, but the free energy profile along the binding path demonstrated that the binding is thermodynamically unfavorable. This explains why the crystal structures of PTE with substrate analogues often exhibit long distances between the phosphoral oxygen and Znβ. The subsequent SN2 reaction plays the key role in the whole process, but controversies exist over the identity of the nucleophilic species, which could be either a hydroxide ion terminally coordinated to Znα or the μ‐hydroxo bridge between the α‐ and β‐metals. Our simulations supported the latter and showed that the rate‐limiting step is the distortion of the bound paraoxon to approach the bridging hydroxide. After this preparation step, the bridging hydroxide ion attacks the phosphorous center and replaces the diethyl phosphate with a low barrier. Thus, a plausible way to engineer PTE with enhanced catalytic activity is to stabilize the deformed paraoxon. Conformational analyses indicate that Trp131 is the closest residue to the phosphoryl oxygen, and mutations to Arg or Gln or even Lys, which can shorten the hydrogen bond distance with the phosphoryl oxygen, could potentially lead to a mutant with enhanced activity for the detoxification of organophosphates.

Exploring the interstitial atom in the FeMo cofactor of nitrogenase: Insights from QM and QM/MM calculations

Density functional theory and combined quantum mechanics and molecular mechanics (QM/MM) calculations have been used to explore structural features of the FeMo cofactor with an interstitial atom X (X = N, C, or O) and its interactions with CO and N 2. Predicted frequencies of the metal-bound CO, QM/MM-optimized geometries, and calculated redox potentials of the FeMo cofactor with different central ligands show that the oxygen atom is the candidate for the interstitial atom. Calculations on the interactions of the FeMo cofactor with CO and N 2 reveal that there is a remarkable dependence of the binding energy on the binding site and the interstitial atom. Generally, the Fe2 site of the FeMo cofactor has stronger interactions with CO and N 2 than Fe6, and both the Fe2 and Fe6 sites in the N-centered and O-centered clusters of the FeMo cofactor can effectively bind N 2 while the coordination of N 2 to the Fe6 site of the C-centered active cluster is unfavorable energetically. Present results indicate that the protein environment is important for computational characterization of the structure of the FeMo cofactor and properties of the metal-bound CO and N 2 are sensitive to the interstitial atom.

Effects of electron attachment on C5'-O5' and C1'-N1 bond cleavages of pyrimidine nucleotides: A theoretical study.

Sugar‐base C1′N1 and phosphate‐sugar C5′O5′ bond breakings of 2′‐deoxycytidine‐5′‐monophosphates (dCMP) and 2′‐deoxythymidine‐5′‐ monophosphates (dTMP) and their radical anions have been explored theoretically at the B3LYP/DZP++ level of theory. Calculations show that the low‐energy electrons attachment to the pyrimidine nucleotides results in remarkable structural and chemical bonding changes. Predicted Gibbs free energies of reaction ΔG for the C5′O5′ bond dissociation process of the radical anions are −14.6 and −11.5 kcal mol−1, respectively, and such dissociation processes may be intrinsically spontaneous in the gas phase. Furthermore, the C5′O5′ bond cleavage processes of the anionic dCMP and dTMP were predicted to have activation energies of 6.9 and 8.0 kcal mol−1 in the gas phase, respectively, much lower than the barriers for the C1′N1 bond breaking process, showing that the CO bond dissociation in DNA single strand breaks is a dominant process as observed experimentally.

Thiol versus hydroxamate as zinc binding group in HDAC inhibition: An Ab initio QM/MM molecular dynamics study

Zinc‐dependent histone deacetylases (HDACs) play a critical role in transcriptional repression and gene silencing, and are among the most attractive targets for the development of new therapeutics against cancer and various other diseases. Two HDAC inhibitors have been approved by FDA as anti‐cancer drugs: one is SAHA whose hydroxamate is directly bound to zinc, the other is FK228 whose active form may use thiol as the zinc binding group. In spite of extensive studies, it remains to be ambiguous regarding how thiol and hydroxamate are bound to the zinc active site of HDACs. In this work, our computational approaches center on Born–Oppenheimer ab initio quantum mechanical/molecular mechanical (QM/MM) molecular dynamics with umbrella sampling, which allow for modeling of the zinc active site with reasonable accuracy while properly including dynamics and effects of protein environment. Meanwhile, an improved short–long effective function (SLEF2) to describe non‐bonded interactions between zinc and other atoms has been employed in initial MM equilibrations. Our ab initio QM/MM MD simulations have confirmed that hydroxamate is neutral when it is bound to HDAC8, and found that thiol is deprotonated when directly bound to zinc in the HDAC active site. By comparing thiol and hydroxamate, our results elucidated the differences in their binding environment in the HDAC active sites, and emphasized the importance of the linker design to achieve more specific binding toward class IIa HDACs.

QM/MM study of catalytic methyl transfer by the N5-glutamine SAM-dependent methyltransferase and its inhibition by the nitrogen analogue of coenzyme

The combined density functional quantum mechanical/molecular mechanical (QM/MM) approach has been used to investigate methyl‐transfer reactions catalyzed by the N5‐glutamine S‐adenosyl‐L‐methionine (SAM)‐dependent methyltransferase (HemK) and the coenzyme‐modified HemK with the replacement of SAM by a nitrogen analogue. Calculations reveal that the catalytic methyl transfer by HemK is an energy‐favored process with an activation barrier of 15.7 kcal/mol and an exothermicity of 12.0 kcal/mol, while the coenzyme‐modified HemK is unable to catalyze the methyl transfer because of a substantial barrier of 20.6 kcal/mol and instability of the product intermediate. The results lend support to the experimental proposal that the nitrogen analogue of the SAM coenzyme should be a practicable inhibitor for the catalytic methyl transfer by HemK. Comparative QM/MM calculations show that the protein environment, especially the residues Asn197 and Pro198 in the active site, plays a pivotal role in stabilizing the transition state and regulating the positioning of reactive groups.