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The structural origin of enzyme cold-adaptation has been the subject of considerable research efforts in recent years. Comparative studies of orthologous mesophilic-psychrophilic enzyme pairs found in nature are an obvious strategy for solving this problem, but they often suffer from relatively low sequence identity of the enzyme pairs. Small bacterial lipases adapted to distinctly different temperatures appear to provide an excellent model system for these types of studies, as they may show a very high degree of sequence conservation. Here, we report the first crystal structures of lipase A from the psychrophilic bacterium Bacillus pumilus , which confirm the high structural similarity to the mesophilic Bacillus subtilis enzyme, as indicated by their 81% sequence identity. We further employ extensive QM/MM calculations to delineate the catalytic reaction path and its energetics. The computational prediction of a rate-limiting deacylation step of the enzymatic ester hydrolysis reaction is verified by stopped-flow experiments, and steady-state kinetics confirms the psychrophilic nature of the B. pumilus enzyme. These results provide a useful benchmark for examining the structural basis of cold-adaptation and should now make it possible to disentangle the effects of the 34 mutations between the two enzymes on catalytic properties and thermal stability.

The structural principles of enzyme cold adaptation are of fundamental interest both for understanding protein evolution and for biotechnological applications. It has become clear in recent years that structural flexibility plays a major role in tuning enzyme activity at low temperatures, which is reflected by characteristic changes in the thermodynamic activation parameters for psychrophilic enzymes, compared to those of mesophilic and thermophilic ones. Hence, increased flexibility of the enzyme surface has been shown to lead to a lower enthalpy and a more negative entropy of activation, which leads to higher activity in the cold. This immediately raises the question of how enzyme oligomerization affects the temperature dependence of catalysis. Here, we address this issue by computer simulations of the catalytic reaction of a cold-adapted bacterial short chain dehydrogenase in different oligomeric states. Reaction free energy profiles are calculated at different temperatures for the tetrameric, dimeric, and monomeric states of the enzyme, and activation parameters are obtained from the corresponding computational Arrhenius plots. The results show that the activation free energy, enthalpy, and entropy are remarkably insensitive to the oligomeric state, leading to the conclusion that assembly of the subunit interfaces does not compromise cold adaptation, even though the mobilities of interfacial residues are indeed affected.

( R )-3-Hydroxybutyrate dehydrogenase (HBDH) catalyzes the NADH-dependent reduction of 3-oxocarboxylates to ( R )-3-hydroxycarboxylates. The active sites of a pair of cold- and warm-adapted HBDHs are identical except for a single residue, yet kinetics evaluated at -5, 0, and 5 °C show a much higher steady-state rate constant ( k cat ) for the cold-adapted than for the warm-adapted HBDH. Intriguingly, single-turnover rate constants ( k STO ) are strikingly similar between the two orthologues. Psychrophilic HBDH primary deuterium kinetic isotope effects on k cat ( D k cat ) and k STO ( D k STO ) decrease at lower temperatures, suggesting more efficient hydride transfer relative to other steps as the temperature decreases. However, mesophilic HBDH D k cat and D k STO are generally temperature-independent. The D k STO data allowed calculation of intrinsic primary deuterium kinetic isotope effects. Intrinsic isotope effects of 4.2 and 3.9 for cold- and warm-adapted HBDH, respectively, at 5 °C, supported by quantum mechanics/molecular mechanics calculations, point to a late transition state for both orthologues. Conversely, intrinsic isotope effects of 5.7 and 3.1 for cold- and warm-adapted HBDH, respectively, at -5 °C indicate the transition state becomes nearly symmetric for the psychrophilic enzyme, but more asymmetric for the mesophilic enzyme. His-to-Asn and Asn-to-His mutations in the psychrophilic and mesophilic HBDH active sites, respectively, swap the single active-site position where these orthologues diverge. At 5 °C, the His-to-Asn mutation in psychrophilic HBDH decreases D k cat to 3.1, suggesting a decrease in transition-state symmetry, while the His-to-Asn mutation in mesophilic HBDH increases D k cat to 4.4, indicating an increase in transition-state symmetry. Hence, temperature adaptation and a single divergent active-site residue may influence transition-state geometry in HBDHs.

The enzyme ( R )-3-hydroxybutyrate dehydrogenase (HBDH) catalyzes the enantioselective reduction of 3-oxocarboxylates to ( R )-3-hydroxycarboxylates, the monomeric precursors of biodegradable polyesters. Despite its application in asymmetric reduction, which prompted several engineering attempts of this enzyme, the order of chemical events in the active site, their contributions to limit the reaction rate, and interactions between the enzyme and non-native 3-oxocarboxylates have not been explored. Here, a combination of kinetic isotope effects, protein crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations were employed to dissect the HBDH mechanism. Initial velocity patterns and primary deuterium kinetic isotope effects establish a steady-state ordered kinetic mechanism for acetoacetate reduction by a psychrophilic and a mesophilic HBDH, where hydride transfer is not rate limiting. Primary deuterium kinetic isotope effects on the reduction of 3-oxovalerate indicate that hydride transfer becomes more rate limiting with this non-native substrate. Solvent and multiple deuterium kinetic isotope effects suggest hydride and proton transfers occur in the same transition state. Crystal structures were solved for both enzymes complexed to NAD + :acetoacetate and NAD + :3-oxovalerate, illustrating the structural basis for the stereochemistry of the 3-hydroxycarboxylate products. QM/MM calculations using the crystal structures as a starting point predicted a higher activation energy for 3-oxovalerate reduction catalyzed by the mesophilic HBDH, in agreement with the higher reaction rate observed experimentally for the psychrophilic orthologue. Both transition states show concerted, albeit not synchronous, proton and hydride transfers to 3-oxovalerate. Setting the MM partial charges to zero results in identical reaction activation energies with both orthologues, suggesting the difference in activation energy between the reactions catalyzed by cold- and warm-adapted HBDHs arises from differential electrostatic stabilization of the transition state. Mutagenesis and phylogenetic analysis reveal the catalytic importance of His150 and Asn145 in the respective orthologues., Competing Interests: The authors declare no competing financial interest., (© 2020 American Chemical Society.)

The existence of temperature optima in enzyme catalysis that occur before protein melting sets in can be described by different types of kinetic models. Such optima cause distinctly curved Arrhenius plots and have, for example, been observed in several cold-adapted enzymes from psychrophilic species. The two main explanations proposed for this behavior either invoke conformational equilibria with inactive substrate-bound states or postulate differences in heat capacity between the reactant and transition states. Herein, we analyze the implications of the different types of kinetic models in terms of apparent activation enthalpies, entropies, and heat capacities, using the catalytic reaction of a cold-adapted α-amylase as a prototypic example. We show that the behavior of these thermodynamic activation parameters is fundamentally different between equilibrium and heat capacity models, and in the α-amylase case, computer simulations have shown the former model to be correct. A few other enzyme-catalyzed reactions are also discussed in this context.

The enzyme-catalyzed degradation of the biogenic amine serotonin is an essential regulatory mechanism of its level in the human organism. In particular, monoamine oxidase A (MAO A) is an important flavoenzyme involved in the metabolism of monoamine neurotransmitters. Despite extensive research efforts, neither the catalytic nor the inhibition mechanisms of MAO enzymes are currently fully understood. In this article, we present the quantum mechanics/molecular mechanics simulation of the rate-limiting step for the serotonin decomposition, which consists of hydride transfer from the serotonin methylene group to the N5 atom of the flavin moiety. Free-energy profiles of the reaction were computed by the empirical valence bond method. Apart from the enzymatic environment, the reference reaction in the gas phase was also simulated, facilitating the estimation of the catalytic effect of the enzyme. The calculated barrier for the enzyme-catalyzed reaction of 14.82 ± 0.81 kcal mol -1 is in good agreement with the experimental value of 16.0 kcal mol -1 , which provides strong evidence for the validity of the proposed hydride-transfer mechanism. Together with additional experimental and computational work, the results presented herein contribute to a deeper understanding of the catalytic mechanism of MAO A and flavoenzymes in general, and in the long run, they should pave the way toward applications in neuropsychiatry.

Cold-adapted enzymes from psychrophilic species show the general characteristics of being more heat labile, and having a different balance between enthalpic and entropic contributions to free energy barrier of the catalyzed reaction compared to mesophilic orthologs. Among cold-adapted enzymes, there are also examples that show an enigmatic inactivation at higher temperatures before unfolding of the protein occurs. Here, we analyze these phenomena by extensive computer simulations of the catalytic reactions of psychrophilic and mesophilic α-amylases. The calculations yield temperature dependent reaction rates in good agreement with experiment, and also elicit the anomalous rate optimum for the cold-adapted enzyme, which occurs about 15 °C below the melting point. This result allows us to examine the structural basis of thermal inactivation, which turns out to be caused by breaking of a specific enzyme-substrate interaction. This type of behaviour is also likely to be relevant for other enzymes displaying such anomalous temperature optima.

Developments in computational chemistry, bioinformatics, and laboratory evolution have facilitated the de novo design and catalytic optimization of enzymes. Besides creating useful catalysts, the generation and iterative improvement of designed enzymes can provide valuable insight into the interplay between the many phenomena that have been suggested to contribute to catalysis. In this work, we follow changes in conformational sampling, electrostatic preorganization, and quantum tunneling along the evolutionary trajectory of a designed Kemp eliminase. We observe that in the Kemp Eliminase KE07, instability of the designed active site leads to the emergence of two additional active site configurations. Evolutionary conformational selection then gradually stabilizes the most efficient configuration, leading to an improved enzyme. This work exemplifies the link between conformational plasticity and evolvability and demonstrates that residues remote from the active sites of enzymes play crucial roles in controlling and shaping the active site for efficient catalysis.

Recent years have seen an explosion of interest in understanding the mechanisms of phosphate ester hydrolysis in biological systems, using a range of computational approaches, each with different advantages and limitations. In this contribution, we present the empirical valence bond (EVB) approach as a powerful tool for modeling biochemical reactivity, using the example of organophosphate hydrolysis by diisopropyl fluorophosphatase as our model reaction. We walk the reader through the protocol for setting up and performing EVB simulations, as well as key technical considerations that need to be taken into account. Finally, we provide examples of the applications of the EVB approach to understanding different experimental observables., (© 2018 Elsevier Inc. All rights reserved.)

Organophosphate hydrolases are proficient catalysts of the breakdown of neurotoxic organophosphates and have great potential as both biotherapeutics for treating acute organophosphate toxicity and as bioremediation agents. However, proficient organophosphatases such as serum paraoxonase 1 (PON1) and the organophosphate-hydrolyzing lactonase SsoPox are unable to hydrolyze bulkyorganophosphates with challenging leaving groups such as diisopropyl fluorophosphate (DFP) or venomous agent X, creating a major challenge for enzyme design. Curiously, despite their mutually exclusive substrate specificities, PON1 and diisopropyl fluorophosphatase (DFPase) have essentially identical active sites and tertiary structures. In the present work, we use empirical valence bond simulations to probe the catalytic mechanism of DFPase as well as temperature, pH, and mutational effects, demonstrating that DFPase and PON1 also likely utilize identical catalytic mechanisms to hydrolyze their respective substrates. However, detailed examination of both static structures and dynamical simulations demonstrates subtle but significant differences in the electrostatic properties and solvent penetration of the two active sites and, most critically, the role of residues that make no direct contact with either substrate in acting as "specificity switches" between the two enzymes. Specifically, we demonstrate that key residues that are structurally and functionally critical for the paraoxonase activity of PON1 prevent it from being able to hydrolyze DFP with its fluoride leaving group. These insights expand our understanding of the drivers of the evolution of divergent substrate specificity in enzymes with identical active sites and guide the future design of organophosphate hydrolases that hydrolyze compounds with challenging leaving groups.

A ground-breaking empirical valence bond study for a soluble transition-metal complex is presented. The full reaction of catalyst monomers approaching and reacting in the Ru V oxidation state were studied. Analysis of the solvation shell in the reactant and along the reaction coordinate revealed that the oxo itself is hydrophobic, which adds a significant driving force to form the dimer. The effect of the solvent on the reaction between the prereactive dimer and the product was small. The solvent seems to lower the barrier for the isoquinoline (isoq) complex while it is increased for pyridines. By comparing the reaction in the gas phase and solution, the proposed π-stacking interaction of the isoq ligands is found to be entirely driven by the water medium., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The tremendous interest in enzymes as biocatalysts has led to extensive work in enzyme engineering, as well as associated methodology development. Here, a new framework for computer-aided directed evolution of enzymes ( CADEE ) is presented which allows a drastic reduction in the time necessary to prepare and analyze in silico semi-automated directed evolution of enzymes. A pedagogical example of the application of CADEE to a real biological system is also presented in order to illustrate the CADEE workflow.

Diverse organophosphate hydrolases have convergently evolved the ability to hydrolyse man-made organophosphates. Thus, these enzymes are attractive model systems for studying the factors shaping enzyme functional evolution. Methyl parathion hydrolase (MPH) is an enzyme from the metallo-β-lactamase superfamily, which hydrolyses a wide range of organophosphate, aryl ester and lactone substrates. In addition, MPH demonstrates metal-ion-dependent selectivity patterns. The origins of this remain unclear, but are linked to open questions about the more general role of metal ions in functional evolution and divergence within enzyme superfamilies. Here, we present detailed mechanistic studies of the paraoxonase and arylesterase activities of MPH complexed with five different transition metal ions, and demonstrate that the hydrolysis reactions proceed via similar pathways and transition states. However, while it is possible to discern a clear structural origin for the selectivity between different substrates, the selectivity between different metal ions appears to lie instead in the distinct electrostatic properties of the metal ions themselves, which causes subtle changes in transition state geometries and metal-metal distances at the transition state rather than significant structural changes in the active site. While subtle, these differences can be significant for shaping the metal-ion-dependent activity patterns observed for this enzyme.This article is part of the themed issue 'Multiscale modelling at the physics-chemistry-biology interface'., (© 2016 The Authors.)

Monoamine oxidases (MAOs) A and B are flavoenzymes responsible for the metabolism of biogenic amines, such as dopamine, serotonin, and noradrenaline (NA), which is why they have been extensively implicated in the etiology and course of various neurodegenerative disorders and, accordingly, used as primary pharmacological targets to treat these debilitating cognitive diseases. The precise chemical mechanism through which MAOs regulate the amine concentration, which is vital for the development of novel inhibitors, is still not unambiguously determined in the literature. In this work, we present atomistic empirical valence bond simulations of the rate-limiting step of the MAO-A-catalyzed NA (norepinephrine) degradation, involving hydride transfer from the substrate α-methylene group to the flavin moiety of the flavin adenine dinucleotide prosthetic group, employing the full dimensionality and thermal fluctuations of the hydrated enzyme, with extensive configurational sampling. We show that MAO-A lowers the free energy of activation by 14.3 kcal mol -1 relative to that of the same reaction in aqueous solution, whereas the calculated activation free energy of ΔG ‡ = 20.3 ± 1.6 kcal mol -1 is found to be in reasonable agreement with the correlated experimental value of 16.5 kcal mol -1 . The results presented here strongly support the fact that both MAO-A and MAO-B isoforms function by the same hydride-transfer mechanism. We also considered a few point mutations of the "aromatic cage" tyrosine residue (Tyr444Phe, Tyr444Leu, Tyr444Trp, Tyr444His, and Tyr444Glu), and the calculated changes in the reaction barriers are in agreement with the experimental values, thus providing further support to the proposed mechanism.

The I335Y point mutation effect on the kinetics of phenylethylamine decomposition catalyzed by monoamine oxidase A was elucidated by means of molecular simulation. The established empirical valence bond methodology was used in conjunction with the free energy perturbation sampling technique and a classical force field representing the state of reactants and products. The methodology allows for the simulation of chemical reactions, in the present case the breaking of the α-C-H bond in a phenylethylamine substrate and the subsequent hydrogen transfer to the flavin cofactor, resulting in the formation of the N-H bond on flavin. The empirical parameters were calibrated against the experimental data for the simulated reaction in a wild type protein and then used for the calculation of the reaction free energy profile in the I335Y mutant. In very good agreement with the measured kinetic data, mutation increases the free energy barrier for the rate limiting step by slightly more than 1 kcal mol(-1) and consequently decreases the rate constant by about an order of magnitude. The magnitude of the computed effect slightly varies with simulation settings, but always remains in reasonable agreement with the experiment. Analysis of trajectories reveals a major change in the interaction between phenyl rings of the substrate and the neighboring Phe352 residue upon the I335Y mutation due to the increased local polarity, leading to an attenuated quadrupole interaction between the rings and destabilization of the transition state. Additionally, the increased local polarity in the mutant allows for a larger number of water molecules to be present near the active site, effectively shielding the catalytic effect of the enzyme and contributing to the increased barrier.

Monoamine oxidases (MAOs) A and B are flavoenzymes responsible for the metabolism of biogenic amines such as dopamine, serotonin and noradrenaline. In this work, we present a comprehensive study of the rate-limiting step of dopamine degradation by MAO B, which consists in the hydride transfer from the methylene group of the substrate to the flavin moiety of the FAD prosthetic group. This article builds on our previous quantum chemical study of the same reaction using a cluster model (Vianello et al., Eur J Org Chem 2012; 7057), but now considering the full dimensionality of the hydrated enzyme with extensive configurational sampling. We show that MAO B is specifically tuned to catalyze the hydride transfer step from the substrate to the flavin moiety of the FAD prosthetic group and that it lowers the activation barrier by 12.3 kcal mol⁻¹ compared to the same reaction in aqueous solution, a rate enhancement of more than nine orders of magnitude. Taking into account the deprotonation of the substrate prior to the hydride transfer reaction, the activation barrier in the enzyme is calculated to be 16.1 kcal mol⁻¹, in excellent agreement with the experimental value of 16.5 kcal mol⁻¹. Additionally, we demonstrate that the protonation state of the active site residue Lys296 does not have an influence on the hydride transfer reaction., (© 2014 Wiley Periodicals, Inc.)

Monoamine oxidases (MAO) A and B are important flavoenzymes involved in the metabolism of amine neurotransmitters. Orru et al. ( J. Neural Transm. 2013 , 120 , 847 - 851 ) recently presented experimental results that have challenged the prevailing assumption that MAO A and MAO B employ an identical catalytic mechanism. We compared the spatial configuration of ionizable groups in both isozymes and estimated the time-averaged electrostatic potential by calculating the pKa values of five active site residues. Superimposition of both experimental structures shows very close overlap and the RMSD in placements of ionizable groups within 16 Å of the reaction center is only 0.847 Å. This similarity is also reflected in the calculated pKa values, where the largest difference between the MAO A and MAO B pKa values was found for residues Tyr188 in MAO B and the corresponding Tyr197 in MAO A assuming 1.23 units. The pKa values for the other four studied residues differ by less than 0.75 units. The results show that the electrostatic preorganizations in both active sites are very similar, supporting the idea that both enzymes work by the same mechanism.

The cationic dummy atom approach provides a powerful nonbonded description for a range of alkaline-earth and transition-metal centers, capturing both structural and electrostatic effects. In this work we refine existing literature parameters for octahedrally coordinated Mn(2+), Zn(2+), Mg(2+), and Ca(2+), as well as providing new parameters for Ni(2+), Co(2+), and Fe(2+). In all the cases, we are able to reproduce both M(2+)-O distances and experimental solvation free energies, which has not been achieved to date for transition metals using any other model. The parameters have also been tested using two different water models and show consistent performance. Therefore, our parameters are easily transferable to any force field that describes nonbonded interactions using Coulomb and Lennard-Jones potentials. Finally, we demonstrate the stability of our parameters in both the human and Escherichia coli variants of the enzyme glyoxalase I as showcase systems, as both enzymes are active with a range of transition metals. The parameters presented in this work provide a valuable resource for the molecular simulation community, as they extend the range of metal ions that can be studied using classical approaches, while also providing a starting point for subsequent parametrization of new metal centers.