2D free-energy surfaces for transfer of the methoxymethyl cation between two water molecules are ... more 2D free-energy surfaces for transfer of the methoxymethyl cation between two water molecules are constructed from molecular dynamics (MD) simulations in which these atoms are treated quantum-mechanically within a box of 1030 classical solvent water molecules at 300 K. This provides a simple model for glycosyl transfer in water. The AM1/TIP3P surfaces with 2D-spline corrections at either MPWB1K/6-31+G(d,p) or MP2/6-31+G(d,p) contain a shallow free-energy well corresponding to an oxacarbenium ion intermediate in a D
Angewandte Chemie (International ed. in English), Jan 12, 2018
The origin of substrate preference in promiscuous enzymes was investigated by enzyme isotope labe... more The origin of substrate preference in promiscuous enzymes was investigated by enzyme isotope labelling of the alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH). At physiological temperature, protein dynamic coupling to the reaction coordinate was insignificant. However, the extent of dynamic coupling was highly substrate-dependent at lower temperatures. For benzyl alcohol, an enzyme isotope effect larger than unity was observed, whereas the enzyme isotope effect was close to unity for isopropanol. Frequency motion analysis on the transition states revealed that residues surrounding the active site undergo substantial displacement during catalysis for sterically bulky alcohols. BsADH prefers smaller substrates, which cause less protein friction along the reaction coordinate and reduced frequencies of dynamic recrossing. This hypothesis allows a prediction of the trend of enzyme isotope effects for a wide variety of substrates.
Phosphoryl transfer reactions are ubiquitous in biology. The reaction mechanism of the phosphoryl... more Phosphoryl transfer reactions are ubiquitous in biology. The reaction mechanism of the phosphorylation of dihydroxyacetone by ATP in aqueous solution has been studied by means of QM/MM simulations in the present paper.
Integrase (IN) is one of the three human immunodeficiency virus type 1 enzymes (HIV-1) essential ... more Integrase (IN) is one of the three human immunodeficiency virus type 1 enzymes (HIV-1) essential for effective viral replication. This viral enzyme is involved in the integration of HIV DNA into host chromosomal DNA. In this work we have carried out molecular dynamics ...
Advances in Protein Chemistry and Structural Biology, 2011
The development of characterization techniques, advanced synthesis methods, as well as molecular ... more The development of characterization techniques, advanced synthesis methods, as well as molecular modeling has transformed the study of systems in a well-established research field. The current research challenges in biocatalysis and biotransformation evolve around enzyme discovery, design, and optimization. How can we find or create enzymes that catalyze important synthetic reactions, even reactions that may not exist in nature? What is the source of enzyme catalytic power? To answer these and other related questions, the standard strategies have evolved from trial-and-error methodologies based on chemical knowledge, accumulated experience, and common sense into a clearly multidisciplinary science that allows one to reach the molecular design of tailor-made enzyme catalysts. This is even more so when one refers to enzyme catalysts, for which the detailed structure and composition are known and can be manipulated to introduce well-defined residues which can be implicated in the chemical rearrangements taking place in the active site. The methods and techniques of theoretical and computational chemistry are becoming more and more important in both understanding the fundamental biological roles of enzymes and facilitating their utilization in biotechnology. Improvement of the catalytic function of enzymes is important from scientific and industrial viewpoints, and to put this fact in the actual perspective as well as the potentialities, we recommend the very recent report of Sanderson [Sanderson, K. (2011). Chemistry: enzyme expertise. Nature 471, 397.]. Great fundamental advances have been made toward the ab initio design of enzyme catalysts based on molecular modeling. This has been based on the molecular mechanistic knowledge of the reactions to be catalyzed, together with the development of advanced synthesis and characterization techniques. The corresponding molecular mechanism can be studied by means of powerful quantum chemical calculations. The catalytic active site can be optimized to improve the transition state analogues (TSA) and to enhance the catalytic activity, even improve the active site to favor a desired direction of some promiscuous enzymes. In this chapter, we give a brief introduction, the state of the art, and future prospects and implications of enzyme design. Current computational tools to assist experimentalists for the design and engineering of proteins with desired catalytic properties are described. The interplay between enzyme design, molecular simulations, and experiments will be presented to emphasize the interdisciplinary nature of this research field. This text highlights the recent advances and examples selected from our laboratory are shown, of how the applications of these tools are a first attempt to de novo design of protein active sites. Identification of neutral/advantageous/deleterious mutation platforms can be exploited to penetrate some of Nature's closely guarded secrets of chemical reactivity. In this chapter, we give a brief introduction, the state of the art, and future prospects and implications of enzyme design. The first part describes briefly how the molecular modeling is carried out. Then, we discuss the requirements of hybrid quantum mechanical/molecular mechanics molecular dynamics (QM/MM MD) simulations, analyzing what are the basis of these theoretical methodologies, how we can use them with a view to its application in the study of enzyme catalysis, and what are the best methodologies for assessing its catalytic potential. In the second part, we focus on some selected examples, taking as a common guide the chorismate to prephenate rearrangement, studying the corresponding molecular mechanism in vacuo, in solution and in an enzyme environment. In addition, examples involving catalytic antibodies (CAs) and promiscuous enzymes will be presented. Finally, a special emphasis is made to provide some hints about the logical evolution that can be anticipated in this research field. Moreover, it helps in understanding the open directions in this area of knowledge and highlights the importance of computational approaches in discovering specific drugs and the impact on the rational design of tailor-made enzymes.
The journal of physical chemistry. B, Jan 22, 2015
The role of protein motions in enzymatic catalysis is the subject of a hot scientific debate. We ... more The role of protein motions in enzymatic catalysis is the subject of a hot scientific debate. We here propose the use of an explicit solvent coordinate to analyze the impact of environmental motions during the reaction process. The example analyzed here is the reaction catalyzed by catechol O-methyltransferase, a methyl transfer reaction from S-adenosylmethionine (SAM) to the nucleophilic oxygen atom of catecholate. This reaction proceeds from a charged reactant to a neutral product, and then a large electrostatic coupling with the environment could be expected. By means of a two-dimensional free energy surface, we show that a large fraction of the environmental motions needed to attain the transition state happens during the first stages of the reaction because most of the environmental motions are slower than changes in the substrate. The incorporation of the solvent coordinate in the definition of the transition state improves the transmission coefficient and the committor histog...
Because of its computational cost, QM/MM simulations are usually carried out using low-quality Ha... more Because of its computational cost, QM/MM simulations are usually carried out using low-quality Hamiltonians, such as semiempirical, which are not always able to provide an accurate potential energy surface. We here propose a simple but efficient way to obtain ...
We use quantum mechanics/molecular mechanics (QM/MM) calculations to estimate the activation free... more We use quantum mechanics/molecular mechanics (QM/MM) calculations to estimate the activation free energy for the chemical reaction catalyzed by catechol O-methyltransferase. While in many cases the activation free energy of a chemical process is directly determined by the potential of mean force associated with a particular reaction coordinate, here we have included several corrections that have been proposed in the literature. These include the free energy change associated with release of the reaction coordinate motion in the reactant state, consideration of the curvilinear nature of the reaction coordinate, and correction due to the classical treatment of molecular vibrations. In addition, since potentials of mean force are usually obtained from low levels of QM theory to describe the quantum subsystem, we have included an interpolated correction term to improve this description at small additional cost. This last correction term has a dramatic effect, improving the agreement between the theoretical predictions and the experimental value, while the other terms considered make only small contributions to this particular reaction.
We present a detailed microscopic study of the dynamics of the Michael addition reaction leading ... more We present a detailed microscopic study of the dynamics of the Michael addition reaction leading from 6'-deoxychalcone to the corresponding flavanone. The reaction dynamics are analyzed for both the uncatalyzed reaction in aqueous solution and the reaction catalyzed by Chalcone Isomerase. By means of rare event simulations of trajectories started at the transition state, we have computed the transmission coefficients, obtaining 0.76 +/- 0.04 and 0.87 +/- 0.03, in water and in the enzyme, respectively. According to these simulations, the Michael addition can be seen as a formation of a new intramolecular carbon-oxygen bond accompanied by a charge transfer essentially taking place from the nucleophilic oxygen to the carbon atom adjacent to the carbonyl group (C (alpha)). As for intermolecular interactions, we find a very significant difference in the evolving solvation pattern of the nucleophilic oxygen in water and in the enzyme. While in the former medium this atom suffers an important desolvation, the enzyme provides, through variations in the distances with some residues and water molecules, an essentially constant electric field on this atom along the reaction progress. Grote-Hynes (GH) theory provides a useful framework to systematically analyze all the couplings between the reaction coordinate and the remaining degrees of freedom. This theory provides transmission coefficients in excellent agreement with the Molecular Dynamics estimations. In contrast, neither the frozen environment approach nor Kramers theory gives results of similar quality, especially in the latter case, where the transmission coefficients are severely underestimated. The (unusual) failure of the frozen environment approach signals the importance of some dynamical motions. Within the context of GH theory, analysis of the friction spectrum obtained in the enzymatic environment, together with normal-mode analysis, is used to identify those motions, of both the substrate and the environment, strongly coupled to the reaction coordinate and to classify them as dynamically active or inactive.
2D free-energy surfaces for transfer of the methoxymethyl cation between two water molecules are ... more 2D free-energy surfaces for transfer of the methoxymethyl cation between two water molecules are constructed from molecular dynamics (MD) simulations in which these atoms are treated quantum-mechanically within a box of 1030 classical solvent water molecules at 300 K. This provides a simple model for glycosyl transfer in water. The AM1/TIP3P surfaces with 2D-spline corrections at either MPWB1K/6-31+G(d,p) or MP2/6-31+G(d,p) contain a shallow free-energy well corresponding to an oxacarbenium ion intermediate in a D
Angewandte Chemie (International ed. in English), Jan 12, 2018
The origin of substrate preference in promiscuous enzymes was investigated by enzyme isotope labe... more The origin of substrate preference in promiscuous enzymes was investigated by enzyme isotope labelling of the alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH). At physiological temperature, protein dynamic coupling to the reaction coordinate was insignificant. However, the extent of dynamic coupling was highly substrate-dependent at lower temperatures. For benzyl alcohol, an enzyme isotope effect larger than unity was observed, whereas the enzyme isotope effect was close to unity for isopropanol. Frequency motion analysis on the transition states revealed that residues surrounding the active site undergo substantial displacement during catalysis for sterically bulky alcohols. BsADH prefers smaller substrates, which cause less protein friction along the reaction coordinate and reduced frequencies of dynamic recrossing. This hypothesis allows a prediction of the trend of enzyme isotope effects for a wide variety of substrates.
Phosphoryl transfer reactions are ubiquitous in biology. The reaction mechanism of the phosphoryl... more Phosphoryl transfer reactions are ubiquitous in biology. The reaction mechanism of the phosphorylation of dihydroxyacetone by ATP in aqueous solution has been studied by means of QM/MM simulations in the present paper.
Integrase (IN) is one of the three human immunodeficiency virus type 1 enzymes (HIV-1) essential ... more Integrase (IN) is one of the three human immunodeficiency virus type 1 enzymes (HIV-1) essential for effective viral replication. This viral enzyme is involved in the integration of HIV DNA into host chromosomal DNA. In this work we have carried out molecular dynamics ...
Advances in Protein Chemistry and Structural Biology, 2011
The development of characterization techniques, advanced synthesis methods, as well as molecular ... more The development of characterization techniques, advanced synthesis methods, as well as molecular modeling has transformed the study of systems in a well-established research field. The current research challenges in biocatalysis and biotransformation evolve around enzyme discovery, design, and optimization. How can we find or create enzymes that catalyze important synthetic reactions, even reactions that may not exist in nature? What is the source of enzyme catalytic power? To answer these and other related questions, the standard strategies have evolved from trial-and-error methodologies based on chemical knowledge, accumulated experience, and common sense into a clearly multidisciplinary science that allows one to reach the molecular design of tailor-made enzyme catalysts. This is even more so when one refers to enzyme catalysts, for which the detailed structure and composition are known and can be manipulated to introduce well-defined residues which can be implicated in the chemical rearrangements taking place in the active site. The methods and techniques of theoretical and computational chemistry are becoming more and more important in both understanding the fundamental biological roles of enzymes and facilitating their utilization in biotechnology. Improvement of the catalytic function of enzymes is important from scientific and industrial viewpoints, and to put this fact in the actual perspective as well as the potentialities, we recommend the very recent report of Sanderson [Sanderson, K. (2011). Chemistry: enzyme expertise. Nature 471, 397.]. Great fundamental advances have been made toward the ab initio design of enzyme catalysts based on molecular modeling. This has been based on the molecular mechanistic knowledge of the reactions to be catalyzed, together with the development of advanced synthesis and characterization techniques. The corresponding molecular mechanism can be studied by means of powerful quantum chemical calculations. The catalytic active site can be optimized to improve the transition state analogues (TSA) and to enhance the catalytic activity, even improve the active site to favor a desired direction of some promiscuous enzymes. In this chapter, we give a brief introduction, the state of the art, and future prospects and implications of enzyme design. Current computational tools to assist experimentalists for the design and engineering of proteins with desired catalytic properties are described. The interplay between enzyme design, molecular simulations, and experiments will be presented to emphasize the interdisciplinary nature of this research field. This text highlights the recent advances and examples selected from our laboratory are shown, of how the applications of these tools are a first attempt to de novo design of protein active sites. Identification of neutral/advantageous/deleterious mutation platforms can be exploited to penetrate some of Nature's closely guarded secrets of chemical reactivity. In this chapter, we give a brief introduction, the state of the art, and future prospects and implications of enzyme design. The first part describes briefly how the molecular modeling is carried out. Then, we discuss the requirements of hybrid quantum mechanical/molecular mechanics molecular dynamics (QM/MM MD) simulations, analyzing what are the basis of these theoretical methodologies, how we can use them with a view to its application in the study of enzyme catalysis, and what are the best methodologies for assessing its catalytic potential. In the second part, we focus on some selected examples, taking as a common guide the chorismate to prephenate rearrangement, studying the corresponding molecular mechanism in vacuo, in solution and in an enzyme environment. In addition, examples involving catalytic antibodies (CAs) and promiscuous enzymes will be presented. Finally, a special emphasis is made to provide some hints about the logical evolution that can be anticipated in this research field. Moreover, it helps in understanding the open directions in this area of knowledge and highlights the importance of computational approaches in discovering specific drugs and the impact on the rational design of tailor-made enzymes.
The journal of physical chemistry. B, Jan 22, 2015
The role of protein motions in enzymatic catalysis is the subject of a hot scientific debate. We ... more The role of protein motions in enzymatic catalysis is the subject of a hot scientific debate. We here propose the use of an explicit solvent coordinate to analyze the impact of environmental motions during the reaction process. The example analyzed here is the reaction catalyzed by catechol O-methyltransferase, a methyl transfer reaction from S-adenosylmethionine (SAM) to the nucleophilic oxygen atom of catecholate. This reaction proceeds from a charged reactant to a neutral product, and then a large electrostatic coupling with the environment could be expected. By means of a two-dimensional free energy surface, we show that a large fraction of the environmental motions needed to attain the transition state happens during the first stages of the reaction because most of the environmental motions are slower than changes in the substrate. The incorporation of the solvent coordinate in the definition of the transition state improves the transmission coefficient and the committor histog...
Because of its computational cost, QM/MM simulations are usually carried out using low-quality Ha... more Because of its computational cost, QM/MM simulations are usually carried out using low-quality Hamiltonians, such as semiempirical, which are not always able to provide an accurate potential energy surface. We here propose a simple but efficient way to obtain ...
We use quantum mechanics/molecular mechanics (QM/MM) calculations to estimate the activation free... more We use quantum mechanics/molecular mechanics (QM/MM) calculations to estimate the activation free energy for the chemical reaction catalyzed by catechol O-methyltransferase. While in many cases the activation free energy of a chemical process is directly determined by the potential of mean force associated with a particular reaction coordinate, here we have included several corrections that have been proposed in the literature. These include the free energy change associated with release of the reaction coordinate motion in the reactant state, consideration of the curvilinear nature of the reaction coordinate, and correction due to the classical treatment of molecular vibrations. In addition, since potentials of mean force are usually obtained from low levels of QM theory to describe the quantum subsystem, we have included an interpolated correction term to improve this description at small additional cost. This last correction term has a dramatic effect, improving the agreement between the theoretical predictions and the experimental value, while the other terms considered make only small contributions to this particular reaction.
We present a detailed microscopic study of the dynamics of the Michael addition reaction leading ... more We present a detailed microscopic study of the dynamics of the Michael addition reaction leading from 6'-deoxychalcone to the corresponding flavanone. The reaction dynamics are analyzed for both the uncatalyzed reaction in aqueous solution and the reaction catalyzed by Chalcone Isomerase. By means of rare event simulations of trajectories started at the transition state, we have computed the transmission coefficients, obtaining 0.76 +/- 0.04 and 0.87 +/- 0.03, in water and in the enzyme, respectively. According to these simulations, the Michael addition can be seen as a formation of a new intramolecular carbon-oxygen bond accompanied by a charge transfer essentially taking place from the nucleophilic oxygen to the carbon atom adjacent to the carbonyl group (C (alpha)). As for intermolecular interactions, we find a very significant difference in the evolving solvation pattern of the nucleophilic oxygen in water and in the enzyme. While in the former medium this atom suffers an important desolvation, the enzyme provides, through variations in the distances with some residues and water molecules, an essentially constant electric field on this atom along the reaction progress. Grote-Hynes (GH) theory provides a useful framework to systematically analyze all the couplings between the reaction coordinate and the remaining degrees of freedom. This theory provides transmission coefficients in excellent agreement with the Molecular Dynamics estimations. In contrast, neither the frozen environment approach nor Kramers theory gives results of similar quality, especially in the latter case, where the transmission coefficients are severely underestimated. The (unusual) failure of the frozen environment approach signals the importance of some dynamical motions. Within the context of GH theory, analysis of the friction spectrum obtained in the enzymatic environment, together with normal-mode analysis, is used to identify those motions, of both the substrate and the environment, strongly coupled to the reaction coordinate and to classify them as dynamically active or inactive.
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