Treatment of NiCl2(dme) and NiBr2(dme) (dme = dimethoxyethane) with 2 equiv of LiOR (OR = OCtBu2Ph) forms the distorted trigonal planar complexes [NiLiX(OR)2(THF)2] (THF = tetrahydrofuran) 5 (X = Cl) and 6 (X = Br). The reaction of CuX2 (X = Cl, Br) with 2 equiv of LiOR affords the Cu(I) product Cu4(OR)4 (7). The same product can be obtained using the Cu(I) starting material CuCl. NMR studies indicated that the reduction of Cu(II) to Cu(I) is accompanied by the oxidation of the alkoxide RO– to form the alkoxy radical RO•, which subsequently forms tert-butyl phenyl ketone by β-scission. Treatment of compounds 1–4 ([M2Li2Cl2(OR)4], M = Cr–Co) with thallium hexafluorophosphate allowed the isolation of the distorted tetrahedral complexes of the form M(OR)2(THF)2 for M = Mn (8), Fe (9), and Co (10). Cyclic voltammetry performed on compounds 8–10 demonstrated irreversible oxidations for all complexes, with the iron complex 9 being the most reducing. Complex 9 shows a reactivity toward PhIO and Ph3SbS to form the corresponding dinuclear iron(III) complexes Fe2(O)(OR)4(THF)2 (11) and Fe2(S)(OR)4(THF)2 (12), respectively. X-ray structural studies were performed, showing that the Fe–O–Fe angle for complex 11 is 176.4(1)° and that the Fe–S–Fe angle for complex 12 is 164.83(3)°.

Most bacteria and all archaea misacylate the tRNAs corresponding to Asn and Gln with Asp and Glu (Asp-tRNAAsn and Glu-tRNAGln).The GatCAB enzyme of most bacteria converts misacylated Glu-tRNAGln to Gln-tRNAGln in order to enable the incorporation of glutamine during protein synthesis. The conversion process involves the intramolecular transfer of ammonia between two spatially separated active sites. This study presents a computational analysis of the two putative intramolecular tunnels that have been suggested to describe the ammonia transfer between the two active sites. Molecular dynamics simulations have been performed for wild-type GatCAB of S. aureus and its mutants: T175(A)V, K88(B)R, E125(B)D, and E125(B)Q. The two tunnels have been analyzed in terms of free energy of ammonia transfer along them. The probability of occurrence of each type of tunnel and the variation of the probability for wild-type GatCAB and its mutants is also discussed.

AlkB is the title enzyme of a family of DNA dealkylases that catalyze the direct oxidative dealkylation of nucleobases. The conventional mechanism for the dealkylation of N1-methyl adenine (1-meA) catalyzed by AlkB after the formation of FeIV–oxo is comprised by a reorientation of the oxo moiety, hydrogen abstraction, OH rebound from the Fe atom to the methyl adduct, and the dissociation of the resulting methoxide to obtain the repaired adenine base and formaldehyde. An alternative pathway with hydroxide as a ligand bound to the iron atom is proposed and investigated by QM/MM simulations. The results show OH– has a small impact on the barriers for the hydrogen abstraction and OH rebound steps. The effects of the enzyme and the OH– ligand on the hydrogen abstraction by the FeIV–oxo moiety are discussed in detail. The new OH rebound step is coupled with a proton transfer to the OH– ligand and results in a novel zwitterion intermediate. This zwitterion structure can also be characterized as Fe–O–C complex and facilitates the formation of formaldehyde. In contrast, for the pathway with H2O bound to iron, the hydroxyl product of the OH rebound step first needs to unbind from the metal center before transferring a proton to Glu136 or other residue/substrate. The consistency between our theoretical results and experimental findings is discussed. This study provides new insights into the oxidative repair mechanism of DNA repair by nonheme FeII and α-ketoglutarate (α-KG) dependent dioxygenases and a possible explanation for the substrate preference of AlkB.

GEM*, a force field that combines Coulomb and Exchange terms calculated with Hermite Gaussians with the polarization, bonded, and modified van der Waals terms from AMOEBA is presented. GEM* is tested on an initial water model fitted at the same level as AMOEBA. The integrals required for the evaluation of the intermolecular Coulomb interactions are efficiently evaluated by means of reciprocal space methods. The GEM* water model is tested by comparing energies and forces for a series of water oligomers and MD simulations. Timings for GEM* compared to AMOEBA are presented and discussed.

•Homology modeling is used to predict the full structure of the ALKBH1 protein.•Disulfide bonds and a zinc finger domain in the structure of ALKBH1 are proposed.•Mutagenesis is performed on residues predicted to form disulfide bonds/zinc finger.•Environment of residues important for lyase function is analyzed.The ability to repair DNA is important for the conservation of genetic information of living organisms. Cells have a number of ways to restore damaged DNA, such as direct DNA repair, base excision repair, and nucleotide excision repair. One of the proteins that can perform direct repair of DNA bases is Escherichia coli AlkB. In humans, there are 9 identified AlkB homologs, including AlkB homolog 1 (ALKBH1). Many of these proteins catalyze the direct oxidative dealkylation of DNA and RNA bases and, as such, have an important role in repairing DNA from damage induced by alkylating agents. In addition to the dealkylase activity, ALKBH1 can also function as an apyrimidinic/apurinic lyase and was proposed to have a distinct lyase active site. To our knowledge, no crystal structure or complete homology model of ALKBH1 protein is available. In this study, we have used homology modeling to predict the structure of ALKBH1 based on AlkB and Duffy-binding-like domain crystal structures as templates. Molecular dynamics simulations were subsequently performed on the predicted structure of ALKBH1. The positions of two disulfide bonds or a zinc-finger motif and a disulfide bond were predicted and the importance of these features was tested by mutagenesis. Possible locations for the lyase active site are proposed based on the analysis of our predicted structures and previous experimental results.

The development of AMOEBA (a multipolar polarizable force field) for imidazolium based ionic liquids is presented. Our parametrization method follows the AMOEBA procedure and introduces the use of QM intermolecular total interactions as well as QM energy decomposition analysis (EDA) to fit individual interaction energy components. The distributed multipoles for the cation and anions have been derived using both the Gaussian distributed multipole analysis (GDMA) and Gaussian electrostatic model-distributed multipole (GEM-DM) methods.1 The intermolecular interactions of a 1,3-dimethylimidazolium [dmim+] cation with various anions, including fluoride [F–], chloride [Cl–], nitrate [NO3–], and tetraflorouborate [BF4–], were studied using quantum chemistry calculations at the MP2/6-311G(d,p) level of theory. Energy decomposition analysis was performed for each pair using the restricted variational space decomposition approach (RVS) at the HF/6-311G(d,p) level. The new force field was validated by running a series of molecular dynamic (MD) simulations and by analyzing thermodynamic and structural properties of these systems. A number of thermodynamic properties obtained from MD simulations were compared with available experimental data. The ionic liquid structure reproduced using the AMOEBA force field is also compared with the data from neutron diffraction experiment and other MD simulations. Employing GEM-DM force fields resulted in a good agreement on liquid densities ρ, enthalpies of vaporization ΔHvap, and diffusion coefficients D± in comparison with conventional force fields.

We demonstrate as a proof of principle the capabilities of a novel hybrid MM′/MM polarizable force field to integrate short-range quantum effects in molecular mechanics (MM) through the use of Gaussian electrostatics. This lead to a further gain in accuracy in the representation of the first coordination shell of metal ions. It uses advanced electrostatics and couples two point dipole polarizable force fields, namely, the Gaussian electrostatic model (GEM), a model based on density fitting, which uses fitted electronic densities to evaluate nonbonded interactions, and SIBFA (sum of interactions between fragments ab initio computed), which resorts to distributed multipoles. To understand the benefits of the use of Gaussian electrostatics, we evaluate first the accuracy of GEM, which is a pure density-based Gaussian electrostatics model on a test Ca(II)–H2O complex. GEM is shown to further improve the agreement of MM polarization with ab initio reference results. Indeed, GEM introduces nonclassical effects by modeling the short-range quantum behavior of electric fields and therefore enables a straightforward (and selective) inclusion of the sole overlap-dependent exchange-polarization repulsive contribution by means of a Gaussian damping function acting on the GEM fields. The S/G-1 scheme is then introduced. Upon limiting the use of Gaussian electrostatics to metal centers only, it is shown to be able to capture the dominant quantum effects at play on the metal coordination sphere. S/G-1 is able to accurately reproduce ab initio total interaction energies within closed-shell metal complexes regarding each individual contribution including the separate contributions of induction, polarization, and charge-transfer. Applications of the method are provided for various systems including the HIV-1 NCp7-Zn(II) metalloprotein. S/G-1 is then extended to heavy metal complexes. Tested on Hg(II) water complexes, S/G-1 is shown to accurately model polarization up to quadrupolar response level. This opens up the possibility of embodying explicit scalar relativistic effects in molecular mechanics thanks to the direct transferability of ab initio pseudopotentials. Therefore, incorporating GEM-like electron density for a metal cation enable the introduction of nonambiguous short-range quantum effects within any point-dipole based polarizable force field without the need of an extensive parametrization.

The combined Electron Localization Funtion (ELF)/ Noncovalent Interaction (NCI) topological analysis (Gillet et al. J. Chem. Theory Comput.2012, 8, 3993) has been extended to enzymatic reaction paths. We applied ELF/NCI to the reactions of DNA polymerase λ and the ε subunit of DNA polymerase III. ELF/NCI is shown to provide insights on the interactions during the evolution of enzymatic reactions including predicting the location of TS from structures located earlier along the reaction coordinate, differential metal coordination, and on barrier differences with two different cations.

AlkB is a bacterial enzyme that catalyzes the dealkylation of alkylated DNA bases. The rate-limiting step is known to be the abstraction of an H atom from the alkyl group on the damaged base by a FeIV-oxo species in the active site. We have used hybrid ab initio quantum mechanical/molecular mechanical methods to study this step in AlkB. Instead of forming an FeIII-oxyl radical from FeIV-oxo near the C–H activation transition state, the reactant is found to be an FeIII-oxyl with an intermediate-spin Fe (S = 3/2) ferromagnetically coupled to the oxyl radical, which we explore in detail using molecular orbital and quantum topological analyses. The minimum energy pathway remains on the quintet surface, but there is a transition between ISFeIII-oxyl and the state with a high-spin Fe (S = 5/2) antiferromagnetically coupled to the oxyl radical. These findings provide clarity for the evolution of the well-known π and σ channels on the quintet surface in the enzyme environment. Additionally, an energy decomposition analysis reveals nine catalytically important residues for the C–H activation step, some of which are conserved in two human homologues. These conserved residues are proposed as targets for experimental mutagenesis studies.

We present the inclusion of distributed multipoles obtained from the Gaussian Electrostatic Model (GEM) into the AMOEBA force field. As a proof of principle, we have reparametrized water and alanine di-peptide. The GEM distributed multipoles (GEM–DM) have been obtained at the same levels of theory as those used for the original AMOEBA parametrization. The use of GEM allows the derivation of the distributed multipoles from the analytical fit to the molecular density or the numerical fit to the molecular electrostatic potential (mESP). In addition, GEM–DM are intrinsically finite of the highest order of the auxiliary basis used for the GEM fit. We also present the fitting of multipoles for the di-methyl imidazolium/chloride (DMIM+–Cl–) ionic liquid pair. Results for intermolecular Coulomb for all test systems show very good agreement. MD simulations for a reparametrized AMOEBA water model with GEM–DM provide results on par with the original AMOEBA force field for a series of bulk properties including liquid density and enthalpy of vaporization. A package for the calculation of GEM Hermite coefficients and derived distributed multipoles using the numerical procedure is also presented and released under the GNU public license.

The Helicobacter pylori (Hp) Asp-tRNAAsn/Glu-tRNAGln amidotransferase (AdT) plays important roles in indirect aminoacylation and translational fidelity. AdT has two active sites, in two separate subunits. Kinetic studies have suggested that interdomain communication occurs between these subunits; however, this mechanism is not well understood. To explore domain–domain communication in AdT, we adapted an assay and optimized it to kinetically characterize the kinase activity of Hp AdT. This assay was applied to the analysis of a series of point mutations at conserved positions throughout the putative AdT ammonia tunnel that connects the two active sites. Several mutations that caused significant decreases in AdT’s kinase activity (reduced by 55–75%) were identified. Mutations at Thr149 (37 Å distal to the GatB kinase active site) and Lys89 (located at the interface of GatA and GatB) were detrimental to AdT’s kinase activity, suggesting that these mutations have disrupted interdomain communication between the two active sites. Models of wild-type AdT, a valine mutation at Thr149, and an arginine mutation at Lys89 were subjected to molecular dynamics simulations. A comparison of wild-type, T149V, and K89R AdT simulation results unmasks 59 common residues that are likely involved in connecting the two active sites.

Recent single-molecule Förster resonance energy transfer studies of DNA polymerase I have led to the proposal of a postinsertion fidelity-checking site. This site is hypothesized to ensure proper base pairing of the newly inserted nucleotide. To help test this hypothesis, we have used energy decomposition, electrostatic free energy response, and noncovalent interaction analysis analyses to identify residues involved in this putative checking site. We have used structures of DNA polymerase I from two different organisms, the Klenow fragment from Escherichia coli and the Bacillus fragment from Bacillus stearothermophilus. Our results point to several residues that show altered interactions for three mispairs compared to the correctly paired DNA dimer. Furthermore, many of these residues are conserved among A family polymerases. The identified residues provide potential targets for mutagenesis studies for investigation of the fidelity-checking site hypothesis.

DNA polymerases require two divalent metal ions in the active site for catalysis. Mg2+ has been confirmed to be the most probable cation utilized by most polymerases in vivo. Other metal ions are either potent mutagens or inhibitors. We used structural and topological analyses based on ab initioQM/MM calculations to study human DNA polymerase λ (Polλ) with different metals in the active site. Our results indicate a slightly longer O3′–Pα distance (∼3.6 Å) for most inhibitor cations compared to the natural and mutagenic metals (∼3.3–3.4 Å). Optimization with a larger basis set for the previously reported transition state (TS) structures (Cisneros et al., DNA Repair, 2008, 7, 1824.) gives barriers of 17.4 kcal mol−1 and 15.1 kcal mol−1 for the Mg2+ and Mn2+ catalyzed reactions respectively. Relying on the key relation between the topological signature of a metal cation and its selectivity within biological systems (de Courcy et al., J. Chem. Theor. Comput., 2010, 6, 1048.) we have performed electron localization function (ELF) topological analyses. These analyses show that all inhibitor and mutagenic metals considered, except Na+, present a “split” of the outer-shell density of the metal. This “splitting” is not observed for the non-mutagenic Mg2+ metal. Population and multipole analyses on the ELF basins reveal that the electronic dipolar and quadrupolar polarization is significantly different with Mg2+ compared to all other cations. Our results shed light at the atomic level on the subtle differences between Mg2+, mutagenic, and inhibitor metals in DNA polymerases. These results provide a correlation between the electronic distribution of the cations in the active site and the possible consequences on DNA synthesis.

E. coli AlkB is a DNA repair enzyme that catalyzes the de-methylation of DNA by means of a non-heme iron and α-keto glutarate as a co-factor. The proposed reaction mechanism can be separated in four stages. The first stage involves the binding of the co-factor and molecular oxygen to the Fe in the active site. This is followed by the formation of a ferryl intermediate in a high-spin state, along with CO2 and succinate. Subsequently, the O atom on the Fe center is reoriented. The last stage comprises the oxidative de-methylation of the base to produce the native DNA base and formaldehyde. This stage also includes the rate limiting step in the reaction. Here, the last stage of the proposed reaction mechanism of AlkB has been studied for a model of the active site with DFT methods. Minimum structures have been calculated for all intermediates along the path in triplet and quintet spin states. Our results point to the quintet states as more stable, in agreement with previously reported calculations. Potential energy barriers have been obtained for all the steps along this last stage in the quintet state. In the first step the oxygen bound to the Fe center of the ferryl intermediate abstracts a hydrogen atom from the methyl moiety. This first step corresponds to the rate limiting step in the reaction. The calculated barrier for this step is 26.7 kcal/mol. The subsequent steps are highly exoergic. This energetic picture is in qualitative agreement with previously reported results. The calculated energy difference between the ferryl intermediate and the final product is −75.7 kcal/mol for a model with succinate in the active site and −49.3 kcal/mol for a model where the succinate is replaced by water. Our calculated mechanism is slightly different than the previously reported one. These results suggest the possibility of more than one mechanism. This is currently under investigation by ab initio QM/MM methods.

•New method to correlate disease SNPs to genes or protein families.•Applied to correlate cancer SNPs to DNA polymerases.•Found 79 new statistically significant SNPs for four cancer phenotypes on DNA polymerases.•Linkage of Polσ and Polλ to prostate and breast cancers, respectively.The advent of complete-genome genotyping across phenotype cohorts has provided a rich source of information for bioinformaticians. However the search for SNPs from this data is generally performed on a study-by-study case without any specific hypothesis of the location for SNPs that are predictive for the phenotype. We have designed a method whereby very large SNP lists (several gigabytes in size), combining several genotyping studies at once, can be sorted and traced back to their ultimate consequence in protein structure. Given a working hypothesis, researchers are able to easily search whole genome genotyping data for SNPs that link genetic locations to phenotypes. This allows a targeted search for correlations between phenotypes and potentially relevant systems, rather than utilizing statistical methods only. HyDn-SNP-S returns results that are less data dense, allowing more thorough analysis, including haplotype analysis. We have applied our method to correlate DNA polymerases to cancer phenotypes using four of the available cancer databases in dbGaP. Logistic regression and derived haplotype analysis indicates that ∼80 SNPs, previously overlooked, are statistically significant. Derived haplotypes from this work link POLL to breast cancer and POLG to prostate cancer with an increase in incidence of 3.01- and 9.6-fold, respectively. Molecular dynamics simulations on wild-type and one of the SNP mutants from the haplotype of POLL provide insights at the atomic level on the functional impact of this cancer related SNP. Furthermore, HyDn-SNP-S has been designed to allow application to any system. The program is available upon request from the authors.

Family I inorganic pyrophosphatases (PPiases) are ubiquitous enzymes that are critical for phosphate metabolism in all domains of life. The detailed catalytic mechanism of these enzymes, including the identity of the general base, is not fully understood. We determined a series of crystal structures of the PPiase from Mycobacterium tuberculosis (Mtb PPiase) bound to catalytic metals, inorganic pyrophosphate (PPi; the reaction substrate) and to one or two inorganic phosphate ions (Pi; the reaction product), ranging in resolution from 1.85 to 3.30 Å. These structures represent a set of major kinetic intermediates in the catalytic turnover pathway for this enzyme and suggest an order of association and dissociation of the divalent metals, the substrate and the two products during the catalytic turnover. The active site of Mtb PPiase exhibits significant structural differences from the well characterized Escherichia coli PPiase in the vicinity of the bound PPi substrate. Prompted by these differences, quantum mechanics/molecular mechanics (QM/MM) analysis yielded an atomic description of the hydrolysis step for Mtb PPiase and, unexpectedly, indicated that Asp89, rather than Asp54 that was proposed for E. coli PPiase, can abstract a proton from a water molecule to activate it for a nucleophilic attack on the PPi substrate. Mutagenesis studies of the key Asp residues of Mtb PPiase supported this mechanism. This combination of structural and computational analyses clarifies our understanding of the mechanism of family I PPiases and has potential utility for rational development of drugs targeting this enzyme.

DNA polymerases require two divalent metal ions in the active site for catalysis. Mg2+ has been confirmed to be the most probable cation utilized by most polymerases in vivo. Other metal ions are either potent mutagens or inhibitors. We used structural and topological analyses based on ab initioQM/MM calculations to study human DNA polymerase λ (Polλ) with different metals in the active site. Our results indicate a slightly longer O3′–Pα distance (∼3.6 Å) for most inhibitor cations compared to the natural and mutagenic metals (∼3.3–3.4 Å). Optimization with a larger basis set for the previously reported transition state (TS) structures (Cisneros et al., DNA Repair, 2008, 7, 1824.) gives barriers of 17.4 kcal mol−1 and 15.1 kcal mol−1 for the Mg2+ and Mn2+ catalyzed reactions respectively. Relying on the key relation between the topological signature of a metal cation and its selectivity within biological systems (de Courcy et al., J. Chem. Theor. Comput., 2010, 6, 1048.) we have performed electron localization function (ELF) topological analyses. These analyses show that all inhibitor and mutagenic metals considered, except Na+, present a “split” of the outer-shell density of the metal. This “splitting” is not observed for the non-mutagenic Mg2+ metal. Population and multipole analyses on the ELF basins reveal that the electronic dipolar and quadrupolar polarization is significantly different with Mg2+ compared to all other cations. Our results shed light at the atomic level on the subtle differences between Mg2+, mutagenic, and inhibitor metals in DNA polymerases. These results provide a correlation between the electronic distribution of the cations in the active site and the possible consequences on DNA synthesis.