Bjørn Olav Brandsdal

In this review we discuss the development of methodology for calculating the temperature dependence and thermodynamic activation parameters for chemical reactions in solution and in enzymes, from computer simulations. We outline how this is done by combining the empirical valence bond method with molecular dynamics free energy simulations. In favorable cases it turns out that such simulations can even capture temperature optima for the catalytic rate. The approach turns out be very useful both for addressing questions regarding the roles of enthalpic and entropic effects in specific enzymes and also for attacking evolutionary problems regarding enzyme adaptation to different temperature regimes. In the latter case, we focus on cold-adaptation of enzymes from psychrophilic species and show how computer simulations have revealed the basic mechanisms behind such adaptation. Understanding these mechanisms also opens up the possibility of designing the temperature dependence, and we highlight a recent example of this.

Cationic, antimicrobial peptides (AMPs) are abundantly present in nature as host-defensive peptides, forming the backbone of the natural defence of many organisms. Their primary interaction with bacterial membranes is thought to be a binding to the outer layer, followed by a disruption of the lipid membrane. Investigating the mode-of-action of AMPs is an ever-growing field, where a plethora of different biophysical experiments are used to investigate this. Here, we explore the experimental limitation in NMR spectroscopy as we migrate towards increasingly realistic but larger membrane model systems with the aim to scout which data we could realistically extract from AMPs interacting with self-aligning nanodiscs. A set of cyclic hexapeptides were used as model compounds, in water, DMSO, and SDS micelles. The quality of isotropic and anisotropic NMR parameters decreased as the result of faster relaxation due to size and dynamics, as well as the need for water suppression and buffer salts. Chemical shifts, scalar couplings, NOE derived distances, and simulated annealing suggested that the backbone conformation of both peptides remained mostly rigid, forming two β-turns, while the sidechains remain flexible in all environments. The difference between Lys or Arg did not change the backbone conformation, even though the two peptides have different MIC concentrations, indicating that amino acid composition is more important than conformation for antimicrobial activity. Theoretical structures in an explicit lipid bilayer were simulated showing similar structures. Pilot experiments on SMA-QA nanodiscs indicated that they are viable as membrane model for investigating the interaction between AMPs and lipid membranes. It was shown that signal can be obtained from bound peptide, but further investigations with isotopically labelled peptides are needed to extract relevant structural data under conditions where the nanodisc is not over-saturated by peptides.

Chorismate mutase (CM) enzymes have long served as model systems for benchmarking new methods and tools in computational chemistry. Despite the enzymes' prominence in the literature, the extent of the roles that activation enthalpy and entropy play in catalyzing the conversion of chorismate to prephenate is still subject to debate. Knowledge of these parameters is a key piece in fully understanding the mechanism of chorismate mutases. Within this study, we utilize EVB/MD free energy perturbation calculations at a range of temperatures, allowing us to extract activation enthalpies and entropies from an Arrhenius plot of activation free energies of the reaction catalyzed by a monofunctional Bacillus subtilis CM and the promiscuous enzyme isochorismate pyruvate lyase of Pseudomonas aeruginosa. In comparison to the uncatalyzed reaction, our results show that both enzyme-catalyzed reactions exhibit a substantial reduction in activation enthalpy, while the effect on activation entropy is relatively minor, demonstrating that enzyme-catalyzed CM reactions are enthalpically driven. Furthermore, we observe that the monofunctional CM from B. subtilis more efficiently catalyzes this reaction than its promiscuous counterpart. This is supported by a structural analysis of the reaction pathway at the transition state, from which we identified key residues explaining the enthalpically driven nature of the reactions and also the difference in efficiencies between the two enzymes.

Chorismate mutases have extensively been used as computational benchmarking systems for enzyme catalysis, yet the roles entropy and enthalpy play in catalysis are still not fully understood. Thus, it is important to better understand these enzymes for potential research or industrial applications. Here, we report the first crystal structure and kinetic characterization of a chorismate mutase from Bacillus pumilus . This enzyme exhibits a high degree of similarity to a known mesophilic chorismate mutase from Bacillus subtilis . Using this crystal structure, we further employ EVB/MD simulations to construct Arrhenius plots, allowing us to extract thermodynamic activation parameters. Overall, this study provides new insights into the structural and functional features of the B. pumilus chorismate mutase and highlights its potential as a valuable enzyme for biocatalytic and biotechnological applications.

Chorismate mutase enzymes have long served as model systems for benchmarking new methods and tools in computational chemistry. Despite the enzymes’ prominence in literature, the extent of the roles activation enthalpy and entropy play in catalyzing the conversion of chorismite to prephenate is still subject to debate. Knowledge of these parameters is a key piece in fully understanding the mechanism of chorismite mutases. Within this study, we utilize EVB/MD free energy perturbations at a range of temperatures, allowing us to extract activation enthalpies and entropies from an Arrhenius plot of reaction free energies of the reaction catalyzed by a monofunctional Bacillus subtilis chorismate mutase and the promiscuous enzyme isochorismate pyruvate lyase of Pseudomonas aeruginosa. In comparison to the uncatalyzed reaction, our results show that both enzyme-catalyzed reactions exhibit a substantial reduction in activation enthalpy, while the effect on activation entropy is relatively minor, demonstrating that enzyme-catalyzed chorismate mutase reactions are enthalpically-driven. Furthermore, we observe that the monofunctional chorismate mutase from B. subtilis more efficiently catalyzes this reaction than its promiscuous counterpart. This is supported by a structural analysis of the reaction pathway at the transition state, from which we identified key residues explaining the enthalpically-driven nature of the reactions, and also the difference in efficiencies between the two enzymes.

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 determination of the temperature dependence of enzyme catalysis has traditionally been a labourious undertaking. We have developed a new approach to the classical Arrhenius parameter estimation by fitting the change in velocity under a gradual change in temperature. The evaluation with a simulated dataset shows that the approach is valid. The approach is demonstrated as a useful tool by characterizing the Bacillus pumilus LipA enzyme. Our results for the lipase show that the enzyme is psychrotolerant, with an activation energy of 15.3 kcal/mol for the chromogenic substrate para-nitrophenyl butyrate. Our results demonstrate that this can produce equivalent curves to the traditional approach while requiring significantly less sample, labour and time. Our method is further validated by characterizing three α-amylases from different species and habitats. The experiments with the α-amylases show that the approach works over a wide range of temperatures and clearly differentiates between psychrophilic, mesophilic and thermophilic enzymes. The methodology is released as an open-source implementation in Python, available online or used locally. This method of determining the activation parameters can make studies of the temperature dependence of enzyme catalysis more widely adapted to understand how enzymes have evolved to function in extreme environments. Moreover, the thermodynamic parameters that are estimated serve as functional validations of the empirical valence bond calculations of enzyme catalysis.

Predicting the effect of single-point mutations on protein stability or protein-ligand binding is a major challenge in computational biology. Free energy calculations constitute the most rigorous approach to this problem, though the estimation of converged values for amino acid mutations is still challenging. To overcome this limitation, we developed tailored protocols to calculate free energy shifts associated to single point mutations. We herein describe QresFEP, which includes an extension of our early protocols to cover all amino acids mutations, and is based on the newest versions of the OPLS-AA forcefields. QresFEP is implemented in an API framework and the graphic interface QGui, for the MD software Q. The complete protocol is benchmarked in several model systems, optimizing a number of variables concerning sampling and the implementation of Zwanzig’s exponential formula and Bennet’s acceptance ratio methods. QresFEP shows an excellent performance on estimating the hydration free energies of amino acid side chain mimics, included their charged analogs. We also examined its performance on a protein-ligand binding problem of pharmaceutical relevance, the antagonism of neuropeptide Y1 G protein-coupled receptor. Here, the calculations show very good agreement with the experimental effect of 16 mutations on the binding of antagonists BIBP3226, in line with our recent applications in this field. Finally, the characterization of 43 mutations of T4-lysozyme reveals the capacity of our protocol to assess variations of the thermal stability of proteins, achieving a similar performance to alternative FEP approaches. In summary, QresFEP is a robust, versatile and user-friendly computational FEP protocol to examine biochemical effects of single-point mutations with high accuracy.

The class I pancreatic elastase from Atlantic salmon is considered to be a cold-adapted enzyme in view of the cold habitat, the reduced thermostability of the enzyme and the fact that it is faster than its mesophilic porcine counterpart at room temperature. However, no experimental characterization of its catalytic properties at lower temperatures have actually been reported. Here we use extensive computer simulations of its catalytic reaction, at different temperatures and with different peptide substrates, to compare its characteristics with those of porcine pancreatic elastase, with which it shares 67% sequence identity. We find that both enzymes have a preference for smaller aliphatic residues at the P1 position, while the reaction rate with phenylalanine at P1 is predicted to be substantially lower. With the former class of substrates the calculated reaction rates for salmon enzyme are consistently higher than those of the porcine ortholog at all temperatures examined and the difference is most pronounced at the lowest temperature. As observed for other cold-adapted enzymes, this is due to redistribution of the activation free energy in terms of enthalpy and entropy and can be linked to differences in the mobility of surface exposed loops in the two enzymes. Such mobility changes are found to be reflected by characteristic sequence conservation patterns in psychrophilic and mesophilic species. Hence, calculations of mutations in a single surface loop show that the temperature dependence of the catalytic reaction is altered in a predictable way.