Computational investigation of cis-1,4-polyisoprene binding to the latex-clearing protein LcpK30.

Latex clearing proteins (Lcps) catalyze the oxidative cleavage of the C = C bonds in cis-1,4-polyisoprene (natural rubber), producing oligomeric compounds that can be repurposed to other materials. The active catalytic site of Lcps is buried inside the protein structure, thus raising the question of how the large hydrophobic rubber chains can access the catalytic center. To improve our understanding of hydrophobic polymeric substrate binding to Lcps and subsequent catalysis, we investigated the interaction of a substrate model containing ten carbon-carbon double bonds with the structurally characterized LcpK30, using multiple computational tools. Prediction of the putative tunnels and cavities in the LcpK30 structure, using CAVER-Pymol plugin 3.0.3, fpocket and Molecular Dynamic (MD) simulations provided valuable insights on how substrate enters from the surface to the buried active site. Two dominant tunnels were discovered that provided feasible routes for substrate binding, and the presence of two hydrophobic pockets was predicted near the heme cofactor. The larger of these pockets is likely to accommodate the substrate and to determine the size distribution of the oligomers. Protein-ligand docking was carried out using GOLD software to predict the conformations and interactions of the substrate within the protein active site. Deeper insight into the protein-substrate interactions, including close-contacts, binding energies and potential cleavage sites in the cis-1,4-polyisoprene, were obtained from MD simulations. Our findings provide further justification that the protein-substrate complexation in LcpK30 is mainly driven by the hydrophobic interactions accompanied by mutual conformational changes of both molecules. Two potential binding modes were identified, with the substrate in either extended or folded conformations. Whilst binding in the extended conformation was most favorable, the folded conformation suggested a preference for cleavage of a central double bond, leading to a preference for oligomers with 5 to 6 C = C bonds. The results provide insight into further enzyme engineering studies to improve catalytic activity and diversify the substrate and product scope of Lcps.

The adaptability of the ion-binding site by the Ag(I)/Cu(I) periplasmic chaperone SilF.

The periplasmic chaperone SilF has been identified as part of an Ag(I) detoxification system in Gram-negative bacteria. Sil proteins also bind Cu(I) but with reported weaker affinity, therefore leading to the designation of a specific detoxification system for Ag(I). Using isothermal titration calorimetry, we show that binding of both ions is not only tighter than previously thought but of very similar affinities. We investigated the structural origins of ion binding using molecular dynamics and QM/MM simulations underpinned by structural and biophysical experiments. The results of this analysis showed that the binding site adapts to accommodate either ion, with key interactions with the solvent in the case of Cu(I). The implications of this are that Gram-negative bacteria do not appear to have evolved a specific Ag(I) efflux system but take advantage of the existing Cu(I) detoxification system. Therefore, there are consequences for how we define a particular metal resistance mechanism and understand its evolution in the environment.

Activation of Glycyl Radical Enzymes─Multiscale Modeling Insights into Catalysis and Radical Control in a Pyruvate Formate-Lyase-Activating Enzyme.

Pyruvate formate-lyase (PFL) is a glycyl radical enzyme (GRE) playing a pivotal role in the metabolism of strict and facultative anaerobes. Its activation is carried out by a PFL-activating enzyme, a member of the radical S-adenosylmethionine (rSAM) superfamily of metalloenzymes, which introduces a glycyl radical into the Gly radical domain of PFL. The activation mechanism is still not fully understood and is structurally based on a complex with a short model peptide of PFL. Here, we present extensive molecular dynamics simulations in combination with quantum mechanics/molecular mechanics (QM/MM)-based kinetic and thermodynamic reaction evaluations of a more complete activation model comprising the 49 amino acid long C-terminus region of PFL. We reveal the benefits and pitfalls of the current activation model, providing evidence that the bound peptide conformation does not resemble the bound protein-protein complex conformation with PFL, with implications for the activation process. Substitution of the central glycine with (S)- and (R)-alanine showed excellent binding of (R)-alanine over unstable binding of (S)-alanine. Radical stabilization calculations indicate that a higher radical stability of the glycyl radical might not be the sole origin of the evolutionary development of GREs. QM/MM-derived radical formation kinetics further demonstrate feasible activation barriers for both peptide and C-terminus activation, demonstrating why the crystalized model peptide system is an excellent inhibitory system for natural activation. This new evidence supports the theory that GREs converged on glycyl radical formation due to the better conformational accessibility of the glycine radical loop, rather than the highest radical stability of the formed peptide radicals.

Dynamic Covalent Self-Assembly of Chloride- and Ion-Pair-Templated Cryptates.

While supramolecular hosts capable of binding and transporting anions and ion pairs are now widely available, self-assembled architectures are still rare, even though they offer an inherent mechanism for the release of the guest ion(s). In this work, we report the dynamic covalent self-assembly of tripodal, urea-based anion cryptates that are held together by two orthoester bridgeheads. These hosts exhibit affinity for anions such as Cl- , Br- or I- in the moderate range that is typically advantageous for applications in membrane transport. In unprecedented experiments, we were able to dissociate the Cs⋅Cl ion pair by simultaneously assembling suitably sized orthoester hosts around the Cs+ and the Cl- ion.

Photodegradation, toxicity and density functional theory study of pharmaceutical metoclopramide and its photoproducts.

Pharmaceuticals as ubiquitous organic pollutants in the aquatic environment represent substances whose knowledge of environmental fate is still limited. One such compound is metoclopramide, whose direct and indirect photolysis and toxicological assessment have been studied for the first time in this study. Experiments were performed under solar radiation, showing metoclopramide as a compound that can easily degrade in different water matrices. The effect of pH-values showed the faster degradation at pH = 7, while the highly alkaline conditions at pH = 11 slowed photolysis. The highest value of quantum yield of metoclopramide photodegradation (ϕ = 43.55·10-4) was obtained at pH = 7. Various organic and inorganic substances (NO3-, Fe(III), HA, Cl-, Br-, HCO3-, SO42-), commonly present in natural water, inhibited the degradation by absorbing light. In all experiments, kinetics followed pseudo-first-order reaction with r2 greater than 0.98. The structures of the photolytic degradation products were tentatively identified, and degradation photoproducts were proposed. The hydroxylation of the aromatic ring and the amino group's dealkylation were two major photoproduct formation mechanisms. Calculated thermochemical quantities are in agreement with the experimentally observed stability of different photoproducts. Reactive sites in metoclopramide were studied with conceptual density functional theory and regions most susceptible to •OH attack were characterized. Metoclopramide and its degradation products were neither genotoxic for bacteria Salmonella typhimurium in the SOS/umuC assay nor acutely toxic for bacteria Vibrio fischeri.

Engineering improved ethylene production: Leveraging systems biology and adaptive laboratory evolution.

Ethylene is a small hydrocarbon gas widely used in the chemical industry. Annual worldwide production currently exceeds 150 million tons, producing considerable amounts of CO2 contributing to climate change. The need for a sustainable alternative is therefore imperative. Ethylene is natively produced by several different microorganisms, including Pseudomonas syringae pv. phaseolicola via a process catalyzed by the ethylene-forming enzyme (EFE), subsequent heterologous expression of EFE has led to ethylene production in non-native bacterial hosts including Escherichia coli and cyanobacteria. However, solubility of EFE and substrate availability remain rate-limiting steps in biological ethylene production. We employed a combination of genome-scale metabolic modelling, continuous fermentation, and protein evolution to enable the accelerated development of a high efficiency ethylene producing E. coli strain, yielding a 49-fold increase in production, the most significant improvement reported to date. Furthermore, we have clearly demonstrated that this increased yield resulted from metabolic adaptations that were uniquely linked to EFE (wild type versus mutant). Our findings provide a novel solution to deregulate metabolic bottlenecks in key pathways, which can be readily applied to address other engineering challenges.

Solvophobically Driven Complexation of Adamantyl Mannoside with ß-Cyclodextrin in Water and Structured Organic Solvents.

Invited for the cover of this issue is Josip Pozar with collaborators from the University of Zagreb. The image depicts the differences in high- and low-temperature water effect on the complexation thermodynamics of adamantyl mannoside with ß-cyclodextrin. Read the full text of the article at 10.1002/chem.202000282.

Solvophobically Driven Complexation of Adamantyl Mannoside with ß-Cyclodextrin in Water and Structured Organic Solvents.

The effects of solvent and temperature on the complexation of adamantyl mannoside with ß-cyclodextrin and 6-O-monotosyl-6-deoxy-ß-cyclodextrin were explored experimentally and by means of molecular dynamics simulations. Efficient binding was observed only in hydrogen-bonded solvents, which indicated solvophobically driven complexation. The stability of the inclusion complex was considerably higher in aqueous media. A pronounced temperature dependence of Δr H○ and Δr S○ , resulting in perfect enthalpy-entropy compensation, was observed in water. The complexation thermodynamics was in line with classical rationale for the hydrophobic effect at lower temperatures and the nonclassical explanation at higher temperatures. This finding linked cyclodextrin complexation thermodynamics with insights regarding the effect of temperature on the hydration water structure. The complexation enthalpies and entropies were weakly dependent on temperature in organic media. The signs of Δr H○ and Δr S○ were in accordance with the nonclassical hydrophobic (solvophobic) effect. The structures of the optimized product corresponded to those deduced spectroscopically, and the calculated and experimentally obtained values of Δr G○ were in very good agreement. This investigation clearly demonstrated that solvophobically driven formation of cyclodextrin complexes could be anticipated in structured solvents in general. However, unlike in water, adamantane and the host cavity behaved solely as structure breakers in the organic media explored so far.

Exploring Reactive Conformations of Coenzyme A during Binding and Unbinding to Pyruvate Formate-Lyase.

Pyruvate formate-lyase (PFL) is a glycyl radical enzyme that converts pyruvate and coenzyme A (CoA) into formate and acetyl-CoA in two half-reactions. Recently, we showed that the acetylation of the PFL active site in the first half-reaction induces subtle conformational changes, leading to the opening of a potential channel for CoA entry. Entry of CoA into the active site is crucial for the second half-reaction, involving the acetyl transfer to CoA, and the completion of the catalytic cycle. Using steered molecular dynamics (SMD) simulations, performed on acetylated and nonacetylated monomeric PFL model systems, we first of all investigate the possible entry/exit pathways of CoA with respect to the active site through the previously identified channel. We then perform umbrella sampling simulations on multiple snapshots from SMD trajectories as well as unrestrained molecular dynamics simulations starting from the final structures obtained from entry SMD, with a view to identifying possible bound states of CoA in the near vicinity of the active site. Detailed study of the unrestrained dissociation processes reveals the presence of stable and reactive bound states of CoA close to the active site, one of which is in an ideal position for triggering the second half-reaction. Examination of the spatial distributions associated with the reactive bound states allows us to discuss the free energy barriers. Umbrella sampling, performed on snapshots from unrestrained dynamics confirms the above findings. The significance of the results for the catalysis are discussed for both acetylated and nonacetylated systems.

Ammonium Complexes of Orthoester Cryptands Are Inherently Dynamic and Adaptive.

Fluxional chemical species such as bullvalene have been a valuable source of inspiration and fundamental insight into the nature of chemical bonds. A supramolecular analogue of bullvalene, i.e., a "fluxional host-guest system", in which the ensemble of a well-defined host and guest is engaged in continuous, degenerate constitutional rearrangements, is still elusive, however. Here, we report experimental and computational evidence for guest-induced dynamic covalent rearrangements in the ammonium complexes of self-assembled orthoester cryptands. This unique behavior is made possible by the ammonium guest playing a dual role: it is sufficiently acidic to initiate dynamic covalent exchange reactions at the orthoester bridgeheads, and as a hydrogen bond donor it acts as a supramolecular template, governing the outcome of a multitude of possible intra- and intermolecular rearrangement reactions. One particularly striking example of inherent dynamic behavior was observed in host-guest complex [NH4+⊂ o-Me2-2.1.1], which spontaneously rearranged into the larger and thermodynamically more stable complex [NH4+⊂ o-Me2-2.2.1], even though this process led to the formation of poor host o-Me2-1.1.1 as a consequence of the excess of one subcomponent (diethylene glycol; "1" in our nomenclature). These inherently adaptive host-guest networks represent a unique platform for exploring the interrelationship between kinetic and thermodynamic stability. For instance, as a result of optimal NH4+ binding, complex [NH4+⊂ o-Me2-2.2.1] was found to be thermodynamically stable (negligible intermolecular rearrangements over weeks), whereas computational studies indicate that the compound is far from kinetically stable ( intramolecular rearrangements).

Functional self-assembled nanovesicles based on ß-cyclodextrin, liposomes and adamantyl guanidines as potential nonviral gene delivery vectors.

Multicomponent self-assembled supramolecular nanovesicles based on an amphiphilic derivative of ß-cyclodextrin and phosphatidylcholine liposomes (PC-liposomes) functionalized with four structurally different adamantyl guanidines were prepared and characterized. Incorporation efficiency of the examined adamantyl guanidines as well as size and surface charge of the prepared supramolecular nanovesicles was determined. Changes in the surface charge of the prepared nanovesicles confirmed that guanidinium groups were exposed on the surface. ITC and 1H NMR spectroscopy complemented by molecular dynamics (MD) simulations were used to elucidate the structural data and stability of the inclusion complexes of ß-cyclodextrin and adamantyl guanidines (AG1-5). The results are consistent and point to a significant contribution of the guanylhydrazone residue to the complexation process for AG1 and AG2 with ß-cyclodextrin. In order to evaluate the potential of the self-assembled supramolecular nanomaterial as a nonviral gene delivery vector, fluorescence correlation spectroscopy was used. It showed that the prepared nanovesicles functionalized with adamantyl guanidines AG1-4 effectively recognize and bind the fluorescently labelled DNA. Furthermore, gel electrophoretic assay confirmed the formation of nanoplexes of functionalized nanovesicles and plasmid DNA. These findings together suggest that the designed supramolecular nanovesicles could be successfully applied as nonviral gene delivery vectors.

The Influence of Chemical Change on Protein Dynamics: A Case Study with Pyruvate Formate-Lyase.

Pyruvate formate-lyase (PFL) catalyzes the reversible conversion of pyruvate and coenzyme A (CoA) into formate and acetyl-CoA in two half-reactions. For the second half-reaction to take place, the S-H group of CoA must enter the active site of the enzyme to retrieve a protein-bound acetyl group. However, CoA is bound at the protein surface, whereas the active site is buried in the protein interior, some 20-30 Šaway. The PFL system was therefore subjected to a series of extensive molecular dynamics simulations (in the µs range) and a host of advanced analysis procedures. Models representing PFL before and after the first half-reaction were used to examine the possible effect of enzyme acetylation. All simulated structures were found to be relatively stable compared to the initial crystal structure. Although the adenine portion of CoA remained predominantly bound at the protein surface, the binding of the S-H group was significantly more labile. A potential entry channel for CoA, which would allow the S-H group to reach the active site, was identified and characterized. The channel was found to be associated with accentuated fluctuations and a higher probability of being in an open state in acetylated systems. This result suggests that the acetylation of the enzyme assumes a prominent functional role, whereby the formation of the acyl intermediate serves to initiate a subtle signaling cascade that influences the protein dynamics and facilitates the entry of the second substrate.

Computational Tale of Two Enzymes: Glycerol Dehydration With or Without B12.

We present a series of QM/MM calculations aimed at understanding the mechanism of the biological dehydration of glycerol. Strikingly and unusually, this process is catalyzed by two different radical enzymes, one of which is a coenzyme-B12-dependent enzyme and the other which is a coenzyme-B12-independent enzyme. We show that glycerol dehydration in the presence of the coenzyme-B12-dependent enzyme proceeds via a 1,2-OH shift, which benefits from a significant catalytic reduction in the barrier. In contrast, the same reaction in the presence of the coenzyme-B12-independent enzyme is unlikely to involve the 1,2-OH shift; instead, a strong preference for direct loss of water from a radical intermediate is indicated. We show that this preference, and ultimately the evolution of such enzymes, is strongly linked with the reactivities of the species responsible for abstracting a hydrogen atom from the substrate. It appears that the hydrogen-reabstraction step involving the product-related radical is fundamental to the mechanistic preference. The unconventional 1,2-OH shift seems to be required to generate a product-related radical of sufficient reactivity to cleave the relatively inactive C-H bond arising from the B12 cofactor. In the absence of B12, it is the relatively weak S-H bond of a cysteine residue that must be homolyzed. Such a transformation is much less demanding, and its inclusion apparently enables a simpler overall dehydration mechanism.

A computational study of the chlorination and hydroxylation of amines by hypochlorous acid.

The reactions of hypochlorous acid (HOCl) with ammonia, (di)methylamine, and heterocyclic amines have been studied computationally using double-hybrid DFT methods (B2PLYP-D and BK-PLYP) and a G3B3 composite scheme. In the gas phase the calculated energy barriers for N- and/or C-hydroxylation are ca. 100 kJ mol(-1) lower than the barrier for N-chlorination of amines. In the model solvent, however, the latter process becomes kinetically more favored. The explicit solvent effects are crucial for determination of the reaction mechanism. The N-chlorination is extremely susceptible to the presence of explicit water molecules, while no beneficial solvation effect has been found for the N- or C-hydroxylation of amines. The origin of the observed solvent effects arises from differential solvation of the respective transition states for chlorine- and oxygen-transfers, respectively. The nature of solvation of the transition state structures has been explored in more detail by classical molecular dynamics (MD) simulation. In agreement with the quantum mechanical approach, the most stable structural motif, which includes the amine, HOCl, and two reactive waters, has been identified during the MD simulation. The inclusion of 5 or 6 explicit water molecules is required to reproduce the experimental barriers for HOCl-induced formation of N-chloramines in an aqueous environment.