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.

Application of a Synthetic Ferredoxin-Inspired [4Fe4S]-Peptide Maquette as the Redox Partner for an [FeFe]-Hydrogenase.

'Bacterial-type' ferredoxins host a cubane [4Fe4S]2+/+ cluster that enables these proteins to mediate electron transfer and facilitate a broad range of biological processes. Peptide maquettes based on the conserved cluster-forming motif have previously been reported and used to model the ferredoxins. Herein we explore the integration of a [4Fe4S]-peptide maquette into a H2 -powered electron transport chain. While routinely formed under anaerobic conditions, we illustrate by electron paramagnetic resonance (EPR) analysis that these maquettes can be reconstituted under aerobic conditions by using photoactivated NADH to reduce the cluster at 240 K. Attempts to tune the redox properties of the iron-sulfur cluster by introducing an Fe-coordinating selenocysteine residue were also explored. To demonstrate the integration of these artificial metalloproteins into a semi-synthetic electron transport chain, we utilize a ferredoxin-inspired [4Fe4S]-peptide maquette as the redox partner in the hydrogenase-mediated oxidation of H2 .

If It Is Hard, It Is Worth Doing: Engineering Radical Enzymes from Anaerobes.

With a pressing need for sustainable chemistries, radical enzymes from anaerobes offer a shortcut for many chemical transformations and deliver highly sought-after functionalizations such as late-stage C-H functionalization, C-C bond formation, and carbon-skeleton rearrangements, among others. The challenges in handling these oxygen-sensitive enzymes are reflected in their limited industrial exploitation, despite what they may deliver. With an influx of structures and mechanistic understanding, the scope for designed radical enzymes to deliver wanted processes becomes ever closer. Combined with new advances in computational methods and workflows for these complex systems, the outlook for an increased use of radical enzymes in future processes is exciting.

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.

Effect of Oriented Electric Fields on Biologically Relevant Iron-Sulfur Clusters: Tuning Redox Reactivity for Catalysis.

Enzyme-based iron-sulfur clusters, exemplified in families such as hydrogenases, nitrogenases, and radical S-adenosylmethionine enzymes, feature in many essential biological processes. The functionality of biological iron-sulfur clusters extends beyond simple electron transfer, relying primarily on the redox activity of the clusters, with a remarkable diversity for different enzymes. The active-site structure and the electrostatic environment in which the cluster resides direct this redox reactivity. Oriented electric fields in enzymatic active sites can be significantly strong, and understanding the extent of their effect on iron-sulfur cluster reactivity can inform first steps toward rationally engineering their reactivity. An extensive systematic density functional theory-based screening approach using OPBE/TZP has afforded a simple electric field-effect representation. The results demonstrate that the orientation of an external electric field of strength 28.8 MV cm-1 at the center of the cluster can have a significant effect on its relative stability in the order of 35 kJ mol-1. This shows clear implications for the reactivity of iron-sulfur clusters in enzymes. The results also demonstrate that the orientation of the electric field can alter the most stable broken-symmetry state, which further has implications on the directionality of initiated electron-transfer reactions. These insights open the path for manipulating the enzymatic redox reactivity of iron-sulfur cluster-containing enzymes by rationally engineering oriented electric fields within the enzymes.

Controlling the outcome of SN2 reactions in ionic liquids: from rational data set design to predictive linear regression models.

Rate constants for a bimolecular nucleophilic substitution (SN2) process in a range of ionic liquids are correlated with calculated parameters associated with the charge localisation on the cation of the ionic liquid (including the molecular electrostatic potential). Simple linear regression models proved effective, though the interdependency of the descriptors needs to be taken into account when considering generality. A series of ionic liquids were then prepared and evaluated as solvents for the same process; this data set was rationally chosen to incorporate homologous series (to evaluate systematic variation) and functionalities not available in the original data set. These new data were used to evaluate and refine the original models, which were expanded to include simple artificial neural networks. Along with showing the importance of an appropriate data set and the perils of overfitting, the work demonstrates that such models can be used to reliably predict ionic liquid solvent effects on an organic process, within the limits of the data set.

Radical Stabilization Energies for Enzyme Engineering: Tackling the Substrate Scope of the Radical Enzyme QueE.

Experimental assessment of catalytic reaction mechanisms and profiles of radical enzymes can be severely challenging due to the reactive nature of the intermediates and sensitivity of cofactors such as iron-sulfur clusters. Here, we present an enzyme-directed computational methodology for the assessment of thermodynamic reaction profiles and screening for radical stabilization energies (RSEs) for the assessment of catalytic turnovers in radical enzymes. We have applied this new screening method to the radical S-adenosylmethione enzyme 7-carboxy-7-deazaguanine synthase (QueE), following a detailed molecular dynamics (MD) analysis that clarifies the role of both specific enzyme residues and bound Mg2+, Ca2+, or Na+. The MD simulations provided the basis for a statistical approach to sample different conformational outcomes. RSE calculation at the M06-2X/6-31+G* level of theory provided the most computationally cost-effective assessment of enzyme-based energies, facilitated by an initial triage using semiempirical methods. The impact of intermolecular interactions on RSE was clearly established, and application to the assessment of potential alternative substrates (focusing on radical clock type rearrangements) proposes a selection of carbon-substituted analogues that would react to afford cyclopropylcarbinyl radical intermediates as candidates for catalytic turnover by QueE.

Proteins in Ionic Liquids: Reactions, Applications, and Futures.

Biopolymer processing and handling is greatly facilitated by the use of ionic liquids, given the increased solubility, and in some cases, structural stability imparted to these molecules. Focussing on proteins, we highlight here not just the key drivers behind protein-ionic liquid interactions that facilitate these functionalities, but address relevant current and potential applications of protein-ionic liquid interactions, including areas of future interest.

Revealing solvent-dependent folding behavior of mycolic acids from Mycobacterium tuberculosis by advanced simulation analysis.

Mycobacterium tuberculosis remains a persistent pathogen, partly due to its lipid rich cell wall, of which mycolic acids (MAs) are a major component. The fluidity and conformational flexibilities of different MAs in the bacterial cell wall significantly influence its properties, function, and observed pathogenicity; thus, a proper conformational description of different MAs in different environments (e.g., in vacuum, in solution, in monolayers) can inform about their potential role in the complex setup of the bacterial cell wall. Previously, we have shown that molecular dynamics (MD) simulations of MA folding in vacuo can be used to characterize MA conformers in seven groupings relating to bending at the functional groups (W, U and Z-conformations). Providing a new OPLS-based forcefield parameterization for the critical cyclopropyl group of MAs and extensive simulations in explicit solvents (TIP4P water, hexane), we now present a more complete picture of MA folding properties together with improved simulation analysis techniques. We show that the 'WUZ' distance-based analysis can be used to pinpoint conformers with hairpin bends at the functional groups, with these conformers constituting only a fraction of accessible conformations. Applying principle component analysis (PCA) and refinement using free energy landscapes (FELs), we are able to discriminate a complete and unique set of conformational preferences for representative alpha-, methoxy- and keto-MAs, with overall preference for folded conformations. A control backbone-MA without any mero-chain functional groups showed significantly less folding in the mero-chain, confirming the role of functionalization in directing folding. Keto-MA showed the highest percentage of WUZ-type conformations and, in particular, a tendency to fold at its alpha-methyl trans-cyclopropane group, in agreement with results from Villeneuve et al. MAs demonstrate similar folding in vacuum and water, with a majority of folded conformations around the W-conformation, although the molecules are more flexible in vacuum than in water. Exchange between conformations, with a disperse distribution that includes unfolded conformers, is common in hexane for all MAs, although with more organization for Keto-MA. Globular, folded conformations are newly defined and may be specifically relevant in biofilms. Graphical abstract Through advanced simulation analysis, including principle component analysis and free energy landscapes, we reveal detailed physical insights into the solvent-dependant folding behavior of mycolic acids from M. tb.

Ion-Reagent Interactions Contributing to Ionic Liquid Solvent Effects on a Condensation Reaction.

Molecular dynamics simulations of solutions of hexan-1-amine or 4-methoxybenzaldehyde in acetonitrile, an ionic liquid/acetonitrile mixture (χIL =0.2), and a number of different (neat) ionic liquids were performed, to further understand the solvent effects on the condensation reaction of these species. This work indicates that, in the presence of an ionic liquid, the amine group of hexan-1-amine is exclusively solvated by the components of the ionic liquid, and not by acetonitrile, and that the anion interacts with the aldehyde group of 4-methoxybenzaldehyde. These interactions showed little dependence on the proportion of the ionic liquid present. When varying the cation of the ionic liquid there were changes in the cation-amine interaction, and 1-butyl-2,3-dimethylimidazolium bis(trifluoromethanesulfonyl)imide ([Bm2 im][N(CF3 SO2 )2 ]) was found to order more than expected about the amine. This ordering is likely the origin of the large rate constant values determined in [Bm2 im][N(CF3 SO2 )2 ] for this condensation reaction and explains an anomaly seen previously. When changing the anion, changes were seen in the interactions between both the cation and anion with hexan-1-amine, and the anion with 4-methoxybenzaldehyde. The differing magnitude of these interactions likely causes subtle changes in the activation parameters for this condensation reaction, and provides an explanation for the anomalous rate constant values previously determined when varying the anion.

Improved NOE fitting for flexible molecules based on molecular mechanics data - a case study with S-adenosylmethionine.

The use of molecular dynamics (MD) calculations to derive relative populations of conformers is highly sensitive to both timescale and parameterisation of the MD. Where these calculations are coupled with NOE data to determine the dynamics of a molecular system, this can present issues if these populations are thus relied upon. We present an approach that refines the highly accurate PANIC NMR methodology combined with clustering approaches to generate conformers, but without restraining the simulations or considering the relative population distributions generated by MD. Combining this structural sampling with NOE fitting, we demonstrate, for S-adenosylmethionine (aqueous solution at pH 7.0), significant improvements are made to the fit of populations to the experimental data, revealing a strong overall preference for the syn conformation of the adenosyl group relative to the ribose ring, but with less discrimination for the conformation of the ribose ring itself.

Radical Reaction Control in the AdoMet Radical Enzyme CDG Synthase (QueE): Consolidate, Destabilize, Accelerate.

Controlling radical intermediates and thus catalysing and directing complex radical reactions is a central feature of S-adensosylmethionine (SAM)-dependent radical enzymes. We report ab initio and DFT calculations highlighting the specific influence of ion complexation, including Mg2+ , identified as a key catalytic component on radical stability and reaction control in 7-carboxy-7-deazaguanine synthase (QueE). Radical stabilisation energies (RSEs) of key intermediates and radical clock-like model systems of the enzyme-catalysed rearrangement of 6-carboxytetrahydropterin (CPH4), reveals a directing role of Mg2+ in destabilising both the substrate-derived radical and corresponding side reactions, with the effect that the experimentally-observed rearrangement becomes dominant over possible alternatives. Importantly, this is achieved with minimal disruption of the thermodynamics of the substrate itself, affording a novel mechanism for an enzyme to both maintain binding potential and accelerate the rearrangement step. Other mono and divalent ions were probed with only dicationic species achieving the necessary radical conformation to facilitate the reaction.

Reactivity of disulfide bonds is markedly affected by structure and environment: implications for protein modification and stability.

Disulfide bonds play a key role in stabilizing protein structures, with disruption strongly associated with loss of protein function and activity. Previous data have suggested that disulfides show only modest reactivity with oxidants. In the current study, we report kinetic data indicating that selected disulfides react extremely rapidly, with a variation of 104 in rate constants. Five-membered ring disulfides are particularly reactive compared with acyclic (linear) disulfides or six-membered rings. Particular disulfides in proteins also show enhanced reactivity. This variation occurs with multiple oxidants and is shown to arise from favorable electrostatic stabilization of the incipient positive charge on the sulfur reaction center by remote groups, or by the neighboring sulfur for conformations in which the orbitals are suitably aligned. Controlling these factors should allow the design of efficient scavengers and high-stability proteins. These data are consistent with selective oxidative damage to particular disulfides, including those in some proteins.

Structural elements of an NRPS cyclization domain and its intermodule docking domain.

Epothilones are thiazole-containing natural products with anticancer activity that are biosynthesized by polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) enzymes EpoA-F. A cyclization domain of EpoB (Cy) assembles the thiazole functionality from an acetyl group and l-cysteine via condensation, cyclization, and dehydration. The PKS carrier protein of EpoA contributes the acetyl moiety, guided by a docking domain, whereas an NRPS EpoB carrier protein contributes l-cysteine. To visualize the structure of a cyclization domain with an accompanying docking domain, we solved a 2.03-Å resolution structure of this bidomain EpoB unit, comprising residues M1-Q497 (62 kDa) of the 160-kDa EpoB protein. We find that the N-terminal docking domain is connected to the V-shaped Cy domain by a 20-residue linker but otherwise makes no contacts to Cy. Molecular dynamic simulations and additional crystal structures reveal a high degree of flexibility for this docking domain, emphasizing the modular nature of the components of PKS-NRPS hybrid systems. These structures further reveal two 20-Å-long channels that run from distant sites on the Cy domain to the active site at the core of the enzyme, allowing two carrier proteins to dock with Cy and deliver their substrates simultaneously. Through mutagenesis and activity assays, catalytic residues N335 and D449 have been identified. Surprisingly, these residues do not map to the location of the conserved HHxxxDG motif in the structurally homologous NRPS condensation (C) domain. Thus, although both C and Cy domains have the same basic fold, their active sites appear distinct.

Three dimensional ink-jet printing of biomaterials using ionic liquids and co-solvents.

1-Ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) and 1-butyl-3-methylimidazolium acetate ([C4C1Im][OAc]) have been used as solvents for the dissolution and ink-jet printing of cellulose from 1.0 to 4.8 wt%, mixed with the co-solvents 1-butanol and DMSO. 1-Butanol and DMSO were used as rheological modifiers to ensure consistent printing, with DMSO in the range of 41-47 wt% producing samples within the printable range of a DIMATIX print-head used (printability parameter < 10) at 55 °C, whilst maintaining cellulose solubility. Regeneration of cellulose from printed samples using water was demonstrated, with the resulting structural changes to the cellulose sample assessed by scanning electron microscopy (SEM) and white light interferometry (WLI). These results indicate the potential of biorenewable materials to be used in the 3D additive manufacture process to generate single-component and composite materials.

Developing energy efficient lignin biomass processing - towards understanding mediator behaviour in ionic liquids.

Environmental concerns have brought attention to the requirement for more efficient and renewable processes for chemicals production. Lignin is the second most abundant natural polymer, and might serve as a sustainable resource for manufacturing fuels and aromatic derivatives for the chemicals industry after being depolymerised. In this work, the mediator 2,2'-azino-bis(3-ethylbenthiazoline-6-sulfonic acid) diammonium salt (ABTS), commonly used with enzyme degradation systems, has been evaluated by means of cyclic voltammetry (CV) for enhancing the oxidation of the non-phenolic lignin model compound veratryl alcohol and three types of lignin (organosolv, Kraft and lignosulfonate) in the ionic liquid 1-ethyl-3-methylimidazolium ethyl sulfate, ([C2mim][C2SO4]). The presence of either veratryl alcohol or organosolv lignin increased the second oxidation peak of ABTS under select conditions, indicating the ABTS-mediated oxidation of these molecules at high potentials in [C2mim][C2SO4]. Furthermore, CV was applied as a quick and efficient way to explore the impact of water in the ABTS-mediated oxidation of both organosolv and lignosulfonate lignin. Higher catalytic efficiencies of ABTS were observed for lignosulfonate solutions either in sodium acetate buffer or when [C2mim][C2SO4] (15 v/v%) was present in the buffer solution, whilst there was no change found in the catalytic efficiency of ABTS in [C2mim][C2SO4]-lignosulfonate mixtures relative to ABTS alone. In contrast, organosolv showed an initial increase in oxidation, followed by a significant decrease on increasing the water content of a [C2mim][C2SO4] solution.

Differential spontaneous folding of mycolic acids from Mycobacterium tuberculosis.

Mycolic acids are structural components of the mycobacterial cell wall that have been implicated in the pathogenicity and drug resistance of certain mycobacterial species. They also offer potential in areas such as rapid serodiagnosis of human and animal tuberculosis. It is increasingly recognized that conformational behavior of mycolic acids is very important in understanding all aspects of their function. Atomistic molecular dynamics simulations, in vacuo, of stereochemically defined Mycobacterium tuberculosis mycolic acids show that they fold spontaneously into reproducible conformational groupings. One of the three characteristic mycolate types, the keto-mycolic acids, behaves very differently from either α-mycolic acids or methoxy-mycolic acids, suggesting a distinct biological role. However, subtle conformational behavioral differences between all the three mycolic acid types indicate that cooperative interplay of individual mycolic acids may be important in the biophysical properties of the mycobacterial cell envelope and therefore in pathogenicity.

Probing the importance of ionic liquid structure: a general ionic liquid effect on an S(N)Ar process.

The effect of a range of ionic liquids, with systematic variations in the cation and anion, on the rate constant of an aromatic substitution process was investigated. Temperature-dependent kinetic data allowed calculation of activation parameters for the process in each solvent. These data demonstrate a generalised ionic liquid effect, with an increase in rate constant observed in each ionic solvent, though the microscopic origins of the rate constant enhancement differ with the nature of the ionic liquid.

Does the cation really matter? The effect of modifying an ionic liquid cation on an SN2 process.

The rate of reaction of a Menschutkin process in a range of ionic liquids with different cations was investigated, with temperature-dependent kinetic data giving access to activation parameters for the process in each solvent. These data, along with molecular dynamics simulations, demonstrate the importance of accessibility of the charged centre on the cation and that the key interactions are of a generalised electrostatic nature.

Enrichment of polyphenol contents and antioxidant activities of Irish brown macroalgae using food-friendly techniques based on polarity and molecular size.

An efficient, food-friendly process for the enrichment of macroalgal phlorotannins from solid-liquid extracts (SLE) of three brown macroalgae, namely Fucus spiralis Linnaeus, Pelvetia canaliculata (Linnaeus) Decaisne & Thuret and Ascophyllum nodosum (Linnaeus) Le Jolis, has been demonstrated. The initial utilisation of molecular weight cut-off (MWCO) dialysis generated fractions of low molecular weight (LMW) (<3.5 kDa) and of high molecular weight (HMW) (3.5-100 kDa and >100 kDa) from cold water, hot water and aqueous ethanolic SLE extracts. An enhancement of the total phenolic content (TPC), radical scavenging abilities (RSA) and ferric reducing antioxidant power (FRAP) in the HMW fractions of 3.5-100 kDa and/or >100 kDa from the cold water and aqueous ethanolic extracts was observed. The initial weak TPC, RSA and FRAP observed in the LMW fractions relative to the HMW fractions were substantially enhanced following a reverse-phase flash chromatography fractionation method. Quadrupole time-of-flight mass spectrometry (Q-Tof-MS) suggests that phlorotannins of varying degrees of phloroglucinol polymerisation are present in LMW fractions of the three brown macroalgal species. The development of a food-friendly process for the extraction and enrichment of phlorotannins from Irish macroalgae is vital to facilitate the use of this valuable resource in future developments of macroalgal-based functional foods.