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Walter Thiel

Publications and source records attributed to Walter Thiel.

At least 19 recordsLinked to original sources
PubMed2007

Learning from directed evolution: Further lessons from theoretical investigations into cooperative mutations in lipase enantioselectivity.

An earlier experimental study, which involved the directed evolution of enantioselective lipase variants from Pseudomonas aeruginosa as catalysts in the hydrolytic kinetic resolution of 2-methyl-decanoic acid p-nitrophenyl ester, provided a mutant with six mutations. Consequently, the selectivity factor was found to increase from E = 1.1 for the wild-type to E = 51 for the best mutant. Only one of the amino acid exchanges in this mutant was found to occur next to the binding pocket, the other mutations being remote. Our previous theoretical analysis with molecular-dynamics simulations helped to unveil the source of enhanced enantioselectivity: a relay mechanism that involves two of the six mutations was shown to induce strong cooperativity. In this investigation, single, double, and triple mutants were constructed and tested as enantioselective catalysts. This study supports our original postulate regarding the relay mechanism, offers further mechanistic insight into the role of individual mutations, and provides mutants that display even higher enantioselectivity (E of up to 64).

Catalysis↗
PubMed2007

Latrunculin analogues with improved biological profiles by "diverted total synthesis": preparation, evaluation, and computational analysis.

Deliberate digression from the blueprint of the total syntheses of latrunculin A (1) and latrunculin B (2) reported in the accompanying paper allowed for the preparation of a focused library of "latrunculin-like" compounds, in which all characteristic structural elements of these macrolides were subject to pertinent molecular editing. Although all previously reported derivatives of 1 and 2 were essentially devoid of any actin-binding capacity, the synthetic compounds presented herein remain fully functional. One of the designer molecules with a relaxed macrocyclic backbone, that is compound 44, even surpasses latrunculin B in its effect on actin while being much easier to prepare. This favorable result highlights the power of "diverted total synthesis" as compared to the much more widely practiced chemical modification of a given lead compound by conventional functional group interconversion. A computational study was carried out to rationalize the observed effects. The analysis of the structure of the binding site occupied by the individual ligands on the G-actin host shows that latrunculin A and 44 both have similar hydrogen-bond network strengths and present similar ligand distortion. In contrast, the H-bond network is weaker for latrunculin B and the distortion of the ligand from its optimum geometry is larger. From this, one may expect that the binding ability follows the order 1 >/= 44 > 2, which is in accord with the experimental data. Furthermore, the biological results provide detailed insights into structure/activity relationships characteristic for the latrunculin family. Thus, it is demonstrated that the highly conserved thiazolidinone ring of the natural products can be replaced by an oxazolidinone moiety, and that inversion of the configuration at C16 (latrunculin B numbering) is also well accommodated. From a purely chemical perspective, this study attests to the maturity of ring-closing alkyne metathesis (RCAM) catalyzed by a molybdenum alkylidyne complex generated in situ, which constitutes a valuable tool for advanced organic synthesis and natural product chemistry.

Actins↗
PubMed2007

The importance of the active site histidine for the activity of epoxide- or aziridine-based inhibitors of cysteine proteases.

In the present study the importance of the active site histidine residue (His) for the activity of epoxide- or aziridine-based cysteine protease inhibitors is examined theoretically. To account for all important effects, QM/MM hybrid approaches are employed which combine quantum mechanical (QM) methods that are necessary to describe bond-breaking and formation processes, with molecular mechanics (MM) methods that incorporate the influence of the protein environment. Using various model systems, the computations exclude a direct proton shift from the active site His residue to the inhibitor, but show that one water molecule is sufficient to establish a very efficient relay system. This relay system allows an easy proton transfer from the active site His residue to the inhibitor and is thus essential for the activity of both types of inhibitors. Differences between the epoxides and the aziridines are discussed, along with some implications for the rational design of optimized inhibitors. The work presented herein represents the first QM/MM study into the mode of action of these important inhibitor classes.

Aziridines↗
PubMed2006

QM/MM study of mechanisms for compound I formation in the catalytic cycle of cytochrome P450cam.

In the catalytic cycle of cytochrome P450cam, after molecular oxygen binds as a ligand to the heme iron atom to yield a ferrous dioxygen complex, there are fast proton transfers that lead to the formation of the active species, Compound I (Cpd I), which are not well understood because they occur so rapidly. In the present work, the conversion of the ferric hydroperoxo complex (Cpd 0) to Cpd I has been investigated by combined quantum-mechanical/molecular-mechanical (QM/MM) calculations. The residues Asp(251) and Glu(366) are considered as proton sources. In mechanism I, a proton is transported to the distal oxygen atom of the hydroperoxo group via a hydrogen bonding network to form protonated Cpd 0 (prot-Cpd0: FeOOH(2)), followed by heterolytic O-O bond cleavage that generates Cpd I and water. Although a local minimum is found for prot-Cpd0 in the Glu(366) channel, it is very high in energy (more than 20 kcal/mol above Cpd 0) and the barriers for its decay are only 3-4 kcal/mol (both toward Cpd 0 and Cpd I). In mechanism II, an initial O-O bond cleavage followed by a concomitant proton and electron transfer yields Cpd I and water. The rate-limiting step in mechanism II is O-O cleavage with a barrier of about 13-14 kcal/mol. According to the QM/MM calculations, the favored low-energy pathway to Cpd I is provided by mechanism II in the Asp(251) channel. Cpd 0 and Cpd I are of similar energies, with a slight preference for Cpd I.

Camphor 5-Monooxygenase↗
PubMed2006

Understanding the enzymatic activity of 4-oxalocrotonate tautomerase and its mutant analogues: a computational study.

The effect of replacing arginine residues (Arg) with citrulline residues (Cit) in the binding site of 4-oxalocrotonate tautomerase (4-OT) was investigated with force field molecular dynamics and hybrid quantum mechanics/molecular mechanics studies. It is found that the Arg61Cit mutation has only minor effects on the k(cat) and K(M) values determined experimentally because of the flexibility of this residue. The decrease in k(cat) and increase in K(M) for the Arg11Cit and Arg39Cit mutations are due to the disruption of the binding site, which arises from repulsive interactions with neighboring residues. The results of this investigation shed new light on the effects of mutations in the binding site of 4-OT and consequently on how the enzyme binds the active substrate.

Arginine↗
PubMed2006

Color tuning in rhodopsins: the mechanism for the spectral shift between bacteriorhodopsin and sensory rhodopsin II.

The mechanism of color tuning in the rhodopsin family of proteins has been studied by comparing the optical properties of the light-driven proton pump bacteriorhodopsin (bR) and the light detector sensory rhodopsin II (sRII). Despite a high structural similarity, the maximal absorption is blue-shifted from 568 nm in bR to 497 nm in sRII. The molecular mechanism of this shift is still a matter of debate, and its clarification sheds light onto the general mechanisms of color tuning in retinal proteins. The calculations employ a combined quantum mechanical/molecular mechanical (QM/MM) technique, using a DFT-based method for ground state properties and the semiempirical OM2/MRCI method and ab initio SORCI method for excited state calculations. The high efficiency of the methodology has allowed us to study a wide variety of aspects including dynamical effects. The absorption shift as well as various mutation experiments and vibrational properties have been successfully reproduced. Our results indicate that several sources contribute to the spectral shift between bR and sRII. The main factors are the counterion region at the extracellular side of retinal and the amino acid composition of the binding pocket. Our analysis allows a distinction and identification of the different effects in detail and leads to a clear picture of the mechanism of color tuning, which is in good agreement with available experimental data.

Bacteriorhodopsins↗
PubMed2006

Analysis of the statistical error in umbrella sampling simulations by umbrella integration.

Umbrella sampling simulations, or biased molecular dynamics, can be used to calculate the free-energy change of a chemical reaction. We investigate the sources of different sampling errors and derive approximate expressions for the statistical errors when using harmonic restraints and umbrella integration analysis. This leads to generally applicable rules for the choice of the bias potential and the sampling parameters. Numerical results for simulations on an analytical model potential are presented for validation. While the derivations are based on umbrella integration analysis, the final error estimate is evaluated from the raw simulation data, and it may therefore be generally applicable as indicated by tests using the weighted histogram analysis method.

Journal Article↗
PubMed2006

The effect of heme environment on the hydrogen abstraction reaction of camphor in P450cam catalysis: a QM/MM study.

The discrepancies between the published QM/MM studies (Schöneboom, J. C.; Cohen, S.; Lin, H.; Shaik, S.; Thiel, W. J. Am. Chem. Soc. 2004, 126, 4017; Guallar, V.; Friesner, R. A. J. Am. Chem. Soc. 2004, 126, 8501) on H-abstraction of camphor in P450cam have largely been resolved. The crystallographic water molecule 903 situated near the oxo atom of Compound I acts as a catalyst for H-abstraction, lowering the barrier by about 4 kcal/mol. Spin density at the A-propionate side chain of heme can occur in the case of incomplete screening but has no major effect on the computed barrier.

Camphor↗
PubMed2006

Thermochemistry of the fluoroformyloxyl radical: a computational study based on coupled cluster theory.

The standard enthalpy of formation of FCO(2) (X (2)B(2)) was determined by a computational approach based on coupled cluster theory [CCSD(T)] with energies extrapolated to the basis-set limit, with additional corrections accounting for core-valence correlation, scalar relativity, spin-orbit coupling, and zero-point vibrational motions. Utilizing a variety of independent reaction schemes, our best estimate is Delta(f)H(o)(0)(FCO(2)) = -86.0 +/- 0.6 kcal mol(-1) [Delta(f)H(o)(298) )(FCO(2)) = -86.7 +/- 0.6 kcal mol(-1)], which is shown to be more accurate than previous theoretical and experimental values. The chosen computational procedure was also applied to HCO (X (2)A'), where we find excellent agreement with experiment, and to FCO (X (2)A'), where we recommend an improved value of Delta(f)H(o)(0)(FCO) = -42.1 +/- 0.5 kcal mol(-1) [ Delta(f)H(o)(298)(FCO) = -42.0 +/- 0.5 kcal mol(-1)]. Further theoretical results concern the C-F bond dissociation energy, electron affinity, ionization energy, first and second excitation energies in FCO(2), fluoride ion affinity of CO(2), and equilibrium geometries of the molecules treated presently. For FCO (X (2)A') we propose an improved equilibrium structure: r(e)(CF) = 132.5(2) pm, r(e)(CO) = 116.7(2) pm, and theta(e)(FCO) = 127.8(2)(o).

Journal Article↗
PubMed2006

Density functional study of enantioselectivity in the 2-methylproline-catalyzed alpha-alkylation of aldehydes.

[structure: see text] An organocatalytic asymmetric alpha-alkylation of aldehydes has recently been shown to provide cyclic aldehydes in high yields and enantioselectivities upon treating substituted acyclic halo-aldehydes with a catalytic amount of 2-methylproline in the presence of 1 equiv of triethylamine. Here, we report a density functional study on the mechanism of this reaction. The crucial step is an intramolecular nucleophilic substitution in the enamine intermediate. The added base accelerates the reaction through the electrostatic activation of the leaving group and affects the stereoselectivity by stabilizing anti and syn transition states to a different extent. On the basis of the computed barriers and transition states, we provide an explanation for the remarkable and unexpected increase in enantioselectivity that is observed when using 2-methylproline instead of proline as the catalyst. Calculated and observed enantiomeric excess values are in good agreement.

Journal Article↗
PubMed2006

Systematic QM/MM investigation of factors that affect the cytochrome P450-catalyzed hydrogen abstraction of camphor.

The hydrogen abstraction reaction of camphor in cytochrome P450(cam) has been investigated in the native enzyme environment by combined quantum mechanical/molecular mechanical (QM/MM) calculations and in the gas phase by density functional calculations. This work has been motivated by contradictory published QM/MM results. In an attempt to pinpoint the origin of these discrepancies, we have systematically studied the factors that may affect the computed barriers, including the QM/MM setup, the optimization procedures, and the choice of QM region, basis set, and protonation states. It is found that the ChemShell and QSite programs used in the published QM/MM calculations yield similar results at given geometries, and that the discrepancies mainly arise from two technical issues (optimization protocols and initial system preparation) that need to be well controlled in QM/MM work. In the course of these systematic investigations, new mechanistic insights have been gained. The crystallographic water 903 placed near the oxo atom of Compound I lowers the hydrogen abstraction barrier by ca. 4 kcal/mol, and thus acts as a catalyst for this reaction. Spin density may appear at the A-propionate side chain of the heme if the carboxylate group is not properly screened, which might be expected to happen during protein dynamics, but not in static equilibrium situations. There is no clear correlation between the computed A-propionate spin density and the hydrogen abstraction barrier, and hence, no support for a previously proposed side-chain mediated transition state stabilization mechanism. Standard QM/MM optimizations yield an A-propionate environment close to the X-ray structure only for protonated Asp297, and not for deprotonated Asp297, but the computed barriers are similar in both cases. An X-ray like A-propionate environment can also be obtained when deprotonated Asp297 is included in the QM region and His355 is singly protonated, but this Compound II-type species with a closed-shell porphyrin ring has a higher hydrogen abstraction barrier and should thus not be mechanistically relevant.

Binding Sites↗
PubMed2005

New features in the catalytic cycle of cytochrome P450 during the formation of compound I from compound 0.

Density functional theory (DFT) is applied to the dark section of the catalytic cycle of the enzyme cytochrome P450, namely, the formation of the active species, Compound I (Cpd I), from the ferric-hydroperoxide species (Cpd 0) by a protonation-assisted mechanism. The chosen 96-atom model includes the key functionalities deduced from experiment: Asp(251), Thr(252), Glu(366), and the water channels that relay the protons. The DFT model calculations show that (a) Cpd I is not formed spontaneously from Cpd 0 by direct protonation, nor is the process very exothermic. The process is virtually thermoneutral and involves a significant barrier such that formation of Cpd I is not facile on this route. (b) Along the protonation pathway, there exists an intermediate, a protonated Cpd 0, which is a potent oxidant since it is a ferric complex of water oxide. Preliminary quantum mechanical/molecular mechanical calculations confirm that Cpd 0 and Cpd I are of similar energy for the chosen model and that protonated Cpd 0 may exist as an unstable intermediate. The paper also addresses the essential role of Thr(252) as a hydrogen-bond acceptor (in accord with mutation studies of the OH group to OMe).

Amino Acids↗
PubMed2005

Bridging the gap between thermodynamic integration and umbrella sampling provides a novel analysis method: "Umbrella integration".

We present a method to analyze biased molecular-dynamics and Monte Carlo simulations, also known as umbrella sampling. In the limiting case of a strong bias, this method is equivalent to thermodynamic integration. It employs only quantities with easily controllable equilibration and greatly reduces the statistical errors compared to the standard weighted histogram analysis method. We show the success of our approach for two examples, one analytic function, and one biological system.

Journal Article↗
PubMed2005

Principal active species of horseradish peroxidase, compound I: a hybrid quantum mechanical/molecular mechanical study.

The active species, Compound I, of horseradish peroxidase (HRP) has been investigated by quantum mechanical/molecular mechanical (QM/MM) calculations using 10 different QM regions. In accord with experimental data, the lowest doublet and quartet states are found to be virtually degenerate, with two unpaired electrons on the FeO moiety and one localized on the porphyrin in an a(2u)-dominant orbital with a minor, but nonnegligible, a(1u) component. The proximal ligand appears to be imidazole rather than imidazolate. The hydrogen-bonding network around the FeO moiety (i.e., Arg38 and His42) has significant influence on the axial bonds and the spin density distribution in the FeO moiety. Including this network in the QM region was found to be essential for reproducing the experimental Mössbauer parameters. The protein environment shapes most of the subtle features of Compound I of HRP.

Horseradish Peroxidase↗
PubMed2005

Insight into enzymatic C-F bond formation from QM and QM/MM calculations.

The C-F bond-forming step in the fluorinase, the only native fluorination enzyme characterized to date, has been studied. The enzyme catalyzes the reaction between S-adenosyl-L-methionine (SAM) and fluoride ions to form 5'-fluoro-5'-deoxyadenosine (5'-FDA) and L-methionine. To obtain an insight into the mechanism of this unusual enzymatic reaction and to elucidate the role of the enzyme in catalysis, we have explored the conformational energetics of SAM and the intrinsic reactivity patterns of SAM and fluoride with DFT (BP86) and continuum solvent methods, before investigating the full enzymatic system with combined DFT/CHARMM calculations. We find that the enzymatic reaction follows an S(N)2 reaction mechanism, concurring with the intrinsic reactivity preferences in solution. The formation of sulfur ylides is thermodynamically strongly disfavored, and an alternative elimination-addition mechanism involving the concerted anti-Markovnikov addition of HF to an enol ether is energetically viable, but kinetically prohibitive. The S(N)2 activation energy is 92 (112) kJ mol(-)(1) in solution, but only 53 (63) kJ mol(-1) in the enzyme, and the reaction energy in the enzyme is -25 (-34) kJ mol(-1) (values in parentheses are B3LYP single-point energies). The fluorinase thus lowers the barrier for C-F bond formation by 39 (49) kJ mol(-)(1). A decomposition analysis shows that the major role of the enzyme is in the preparation and positioning of the substrates.

Journal Article↗
PubMed2005

Combination of computational prescreening and experimental library construction can accelerate enzyme optimization by directed evolution.

Chiral compounds can be produced efficiently by using biocatalysts. However, wild-type enzymes often do not meet the requirements of a production process, making optimization by rational design or directed evolution necessary. Here, we studied the lipase-catalyzed hydrolysis of the model substrate 1-(2-naphthyl)ethyl acetate both theoretically and experimentally. We found that a computational equivalent of alanine scanning mutagenesis based on QM/MM methodology can be applied to identify amino acid positions important for the activity of the enzyme. The theoretical results are consistent with concomitant experimental work using complete saturation mutagenesis and high-throughput screening of the target biocatalyst, a lipase from Bacillus subtilis. Both QM/MM-based calculations and molecular biology experiments identify histidine 76 as a residue that strongly affects the catalytic activity. The experiments demonstrate its important influence on enantioselectivity.

Amino Acid Substitution↗
PubMed2005

Potential-energy surface for the electronic ground state of NH3 up to 20,000 cm-1 above equilibrium.

Ab initio coupled cluster calculations with single and double substitutions and a perturbative treatment of connected triple excitations [CCSD(T)] with the augmented correlation-consistent polarized valence triple-zeta aug-cc-pVTZ basis at 51 816 geometries provide a six-dimensional potential-energy surface for the electronic ground state of NH3. At 3814 selected geometries, CBS+ energies are obtained by extrapolating the CCSD(T) results for the aug-cc-pVXZ(X=T,Q,5) basis sets to the complete basis set (CBS) limit and adding corrections for core-valence correlation and relativistic effects. CBS** ab initio energies are generated at 51,816 geometries by an empirical extrapolation of the CCSD(T)/aug-cc-pVTZ results to the CBS+ limit. They cover the energy region up to 20,000 cm-1 above equilibrium. Parametrized analytical functions are fitted through the ab initio points. For these analytical surfaces, vibrational term values and transition moments are calculated by means of a variational program employing a kinetic-energy operator expressed in the Eckart-Sayvetz frame. Comparisons against experiment are used to assess the quality of the generated potential-energy surfaces. A "spectroscopic" potential-energy surface of NH3 is determined by a slight empirical adjustment of the ab initio potential to the experimental vibrational term values. Variational calculations on this refined surface yield rms deviations from experiment of 0.8 cm-1 for 24 inversion splittings and 0.4 (3.0) cm-1 for 34 (51) vibrational term values up to 6100 (10,300) cm-1.

Journal Article↗