Sergio Marti | Universitat Jaume I de Castelló (original) (raw)

Papers by Sergio Marti

Biochemistry, 2014

Because of the increasing resistance of malaria parasites to antimalarial drugs, the lack of high... more Because of the increasing resistance of malaria parasites to antimalarial drugs, the lack of highly effective vaccines, and an inadequate control of mosquito vectors, the problem is growing, especially in the developing world. New approaches to drug development are consequently required. One of the proteases involved in the degradation of human hemoglobin is named falcipain-2 (FP2), which has emerged as a promising target for the development of novel antimalarial drugs. However, very little is known about the inhibition of FP2. In this paper, the inhibition of FP2 by the epoxysuccinate E64 has been studied by molecular dynamics (MD) simulations using hybrid AM1d/MM and M06-2X/ MM potentials to obtain a complete picture of the possible free energy reaction paths. A thorough analysis of the reaction mechanism has been conducted to understand the inhibition of FP2 by E64. According to our results, the irreversible attack of Cys42 on E64 can take place on both carbon atoms of the epoxy ring because both processes present similar barriers. While the attack on the C2 atom presents a slightly smaller barrier (12.3 vs 13.6 kcal mol −1 ), the inhibitor−protein complex derived from the attack on C3 appears to be much more stabilized. In contrast to previous hypotheses, our results suggest that residues such as Gln171, Asp170, Gln36, Trp43, Asn81, and even His174 would be anchoring the inhibitor in a proper orientation for the reaction to take place. These results may be useful for the rational design of new compounds with higher inhibitory activity. . performed the bulk of computational simulations. S.M. wrote the codes and guided some of the calculations. S.M., S.F., and V.M. designed the research. K.A., S.F., and V.M. cowrote the first version of the manuscript. All the authors analyzed data, and V.M. wrote the final version of the manuscript.

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.

Theoretical Chemistry Accounts, 2011

Salicylate synthase from Mycobacterium tuberculosis, MbtI, initiates the biosynthesis of sideroph... more Salicylate synthase from Mycobacterium tuberculosis, MbtI, initiates the biosynthesis of siderophores by converting chorismate to salicylate. Nevertheless, three more distinct activities for wild-type MbtI have been detected in vitro: isochorismate synthase, isochorismate pyruvate lyase, and chorismate mutase. In this work, hybrid Quantum Mechanics/Molecular Mechanics methods have been used to get the first simulation of the chorismate mutase activity of MbtI. The results show how the reaction proceeds by means of a [3,3] sigmatropic rearrangement with free energy barrier in very good agreement with experiments.

Physical Chemistry Chemical Physics, 2012

Salicylate synthase from Mycobacterium tuberculosis, MbtI, is a highly promiscuous Mg 2+ dependen... more Salicylate synthase from Mycobacterium tuberculosis, MbtI, is a highly promiscuous Mg 2+ dependent enzyme with up to four distinct activities detected in vitro: isochorismate synthase (IS), isochorismate pyruvate lyase (IPL), salicylate synthase (SS) and chorismate mutase (CM). In this paper, Molecular Dynamic (MD) simulations employing hybrid quantum mechanics/molecular mechanics (QM/MM) potentials have been carried out to get a detailed knowledge of the IS and the IPL activities at the molecular level. According to our simulations, the architecture of the MbtI active site allows catalyzing the two reactions: the isochorismate formation, by means of a stepwise mechanism, and the salicylate production from isochorismate, that appears to be pericyclic in nature. Findings also explain the role of the magnesium cation and the pH dependence activity experimentally observed in MbtI. Mg 2+ would be polarizing and pre-organizing the substrate and active site, as well as shifting the pK a values of key active site residues. w Electronic supplementary information (ESI) available: to S7 containing X-ray structures of Irp9 (2FN1) and MbtI (2G5F), 2D-PMFs of reaction corresponding to the IS activity of MbtI corrected at the M06-2X level and representative structures of the substrate in chorismate, intermediate, isochorismate and transition states located in the active site of the MbtI, 2D-PMF of reaction corresponding to the IPL activity of MbtI corrected at the M06-2X level and representative structures of the substrate in the isochorismate, transition state and products (salicylate and pyruvate), representative snapshots of the K205Q mutated enzyme compared to the wild-type and time dependent RMSD during equilibration of the system. See

Journal of computational chemistry, Jan 19, 2015

CYP19A1 aromatase is a member of the Cytochrome P450 family of hemeproteins, and is the enzyme re... more CYP19A1 aromatase is a member of the Cytochrome P450 family of hemeproteins, and is the enzyme responsible for the final step of the androgens conversion into the corresponding estrogens, via a three-step oxidative process. For this reason, the inhibition of this enzyme plays an important role in the treatment of hormone-dependent breast cancer. The first catalytic subcycle, corresponding to the hydroxilation of androstenedione, has been proposed to occur through a first hydrogen abstraction and a subsequent oxygen rebound step. In present work, we have studied the mechanism of the first catalytic subcycle by means of hybrid quantum mechanics/molecular mechanics methods. The inclusion of the protein flexibility has been achieved by means of Free Energy Perturbation techniques, giving rise to a free energy of activation for the hydrogen abstraction step of 13.5 kcal/mol. The subsequent oxygen rebound step, characterized by a small free energy barrier (1.5 kcal/mol), leads to the hydr...

ACS catalysis, Jan 7, 2015

In the last decade L-Lactate Dehydrogenase (LDH) has become an extremely useful marker in both cl... more In the last decade L-Lactate Dehydrogenase (LDH) has become an extremely useful marker in both clinical diagnosis and in monitoring the course of many human diseases. It has been assumed from the 80s that the full catalytic process of LDH starts with the binding of the cofactor and the substrate followed by the enclosure of the active site by a mobile loop of the protein before the reaction to take place. In this paper we show that the chemical step of the LDH catalyzed reaction can proceed within the open loop conformation, and the different reactivity of the different protein conformations would be in agreement with the broad range of rate constants measured in single molecule spectrometry studies. Starting from a recently solved X-ray diffraction structure that presented an open loop conformation in two of the four chains of the tetramer, QM/MM free energy surfaces have been obtained at different levels of theory. Depending on the level of theory used to describe the electronic s...

Journal of Physical Chemistry B, 2008

We discuss the role of the protein in controlling the absorption spectra of photoactive yellow pr... more We discuss the role of the protein in controlling the absorption spectra of photoactive yellow protein (PYP), the archetype xanthopsin photoreceptor, using quantum mechanics/molecular mechanics (QM/MM) methods based on ab initio multireference perturbation theory, combined with molecular dynamics (MD) simulations. It is shown that in order to get results in agreement with the experimental data, it is necessary to use a model that allows for a proper relaxation of the whole system and treats the states involved in the electronic spectrum in a balanced way, avoiding biased results due to the effect of nonrepresentative electrostatic interactions on the chromophore.

The journal of physical chemistry. A, Jan 19, 2006

Catalytic antibodies are very interesting not only because of the rate enhancement of the reactio... more Catalytic antibodies are very interesting not only because of the rate enhancement of the reactions that they catalyze but also because of the selectivities they can achieve that are sometimes not present in natural enzyme processes. We have selected the study of the stereoselectivity of the matured AZ28 that catalyzes an oxy-Cope rearrangement. For this particular case, the presence of a chiral center in the substrate provokes the existence of two different enantiomers, R and S. Furthermore, it is also possible to locate two different orientations for the hydroxyl group in the central ring of the substrate in the transition state, equatorial and axial, rendering two different conformers. In this paper we present the free energy profiles obtained for different substrate isomers in the cavity created by the matured catalytic antibody. Our simulations have reproduced the stereoselectivity of the matured AZ28, differentiating between the axial or equatorial orientations and preferentia...

The Journal of Physical Chemistry B, 2011

Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations were performed to ... more Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations were performed to investigate the methylation of 6-mercaptopurine catalyzed by thiopurine S-methyltransferase. Several setups with different tautomeric forms and orientations of the substrate were considered. It is found that, with the orientation in chain A of the X-ray structure, the substrate can form an ideal near-attack configuration for the methylation reaction, which may take place after the deprotonation of the substrate by the conserved residue Asp23 through a water chain. The potential of mean force (PMF) of the methyl-transfer step for the most favorable pathway is 19.6 kcal/ mol, which is in good agreement with the available experimental rate constant data.

Organic & Biomolecular Chemistry, 2003

Kinetic isotope effects have been computed for the Claisen rearrangement of chorismate to prephen... more Kinetic isotope effects have been computed for the Claisen rearrangement of chorismate to prephenate in aqueous solution and in the active site of chorismate mutase from B. subtilus. These included primary 13C and 18O and secondary 3H effects for substitutions at the bond-making and bond-breaking positions. The initial structures of the putative stationary points on the potential energy surface, required for the calculations of isotope effects using the CAMVIB/CAMISO programs, have been selected from hybrid QM/MM molecular dynamical simulations using the DYNAMO program. Refinement of the reactant complex and transition-state structures has been carried out by means of AM1/CHARMM24/TIP3P calculations using the GRACE program, with full gradient relaxation of the position of > 5200 atoms for the enzymic simulations, and with a box containing 711 water molecules for the corresponding reaction in aqueous solution. Comparison of these results, and of gas phase calculations, with experimental data has shown that the chemical rearrangement is largely rate-determining for the enzyme mechanism. Inclusion of the chorismate conformational pre-equilibrium step in the modelled kinetic scheme leads to better agreement between recent experimental data and theoretical predictions. These results provide new information on an important enzymatic transformation, and the key factors responsible for the kinetics of its molecular mechanism are clarified. Treatment of the enzyme and/or solvent environment by means of a large and flexible model is absolutely essential for prediction of kinetic isotope effects.

Nature Chemistry, 2013

Conformational changes are known to be able to drive an enzyme through its catalytic cycle, allow... more Conformational changes are known to be able to drive an enzyme through its catalytic cycle, allowing, for example, substrate binding or product release. However, the influence of protein motions on the chemical step is a controversial issue. One proposal is that the simple equilibrium fluctuations incorporated into transition-state theory are insufficient to account for the catalytic effect of enzymes and that protein motions should be treated dynamically. Here, we propose the use of free-energy surfaces, obtained as a function of both a chemical coordinate and an environmental coordinate, as an efficient way to elucidate the role of protein structure and motions during the reaction. We show that the structure of the protein provides an adequate environment for the progress of the reaction, although a certain degree of flexibility is needed to attain the full catalytic effect. However, these motions do not introduce significant dynamical corrections to the rate constant and can be described as equilibrium fluctuations.

Journal of the American Chemical Society, 2009

The isochorismate pyruvate lyase (IPL) from Pseudomonas aeruginosa, designated as PchB, catalyzes... more The isochorismate pyruvate lyase (IPL) from Pseudomonas aeruginosa, designated as PchB, catalyzes the transformation of isochorismate into pyruvate and salicylate, but it also catalyzes the rearrangement of chorismate into prephenate, suggesting that both reactions may proceed by a pericyclic mechanism. In this work, molecular dynamics simulations employing hybrid quantum mechanics/molecular mechanics methods have been carried out to get a detailed knowledge of the reaction mechanism of PchB. The results provide a theoretical rate constant enhancement by comparison with the reaction in solution, in agreement with the experimental data, and confirm the pericyclic nature of the reaction mechanism. The robustness of this promiscuous enzyme has been checked by considering the impact of Ala37Ile mutation, previously proposed by us to improve the secondary chorismate mutase (CM) activity. The effect of this mutation, which was shown to increase the rate constant for the CM activity by a factor of 10(3), also increases the IPL catalytic efficiency, although only by a factor of 6.

Journal of the American Chemical Society, 2006

In some enzymatic systems large conformational changes are coupled to the chemical step, in such ... more In some enzymatic systems large conformational changes are coupled to the chemical step, in such a way that dispersion of rate constants can be observed in single-molecule experiments, each corresponding to the reaction from a different reactant valley. Under this perspective here we present a computational study of pyruvate to lactate transformation catalyzed by lactate dehydrogenase. The reaction consists of a hydride transfer and a proton transfer that seem to take place concertedly. The degree of asynchronicity and the energy barrier depend on the particular starting reactant valley. In order to estimate rate constants we used a free energy perturbation technique adapted to follow the intrinsic reaction coordinate for several possible reaction paths. Tunneling effects are also obtained with a slightly modified version of the ensemble-averaged variational transition state theory with multidimensional tunneling contributions. According to our results the closure of the active site by means of a flexible loop can lead to the formation of different reactant complexes displaying different features in the disposition of some key residues (such as Arg109), interactions with the substrate and number of water molecules in the active site. The chemical step of the reaction takes place with a different reaction rate from each of these complexes. Finally, primary kinetic isotope effects for replacement of the transferring hydrogen of the cofactor with a deuteride are in good agreement with experimental observations, thus validating our methodology.

Journal of the American Chemical Society, 2004

In this work we present a detailed analysis of the activation free energies and averaged interact... more In this work we present a detailed analysis of the activation free energies and averaged interactions for the Claisen and Cope rearrangements of chorismate and carbachorismate catalyzed by Bacillus subtilis chorismate mutase (BsCM) using quantum mechanics/molecular mechanics (QM/MM) simulation methods. In gas phase, both reactions are described as concerted processes, with the activation free energy for carbachorismate being about 10-15 kcal mol -1 larger than for chorismate, at the AM1 and B3LYP/6-31G* levels. Aqueous solution and BsCM active site environments reduce the free energy barriers for both reactions, due to the fact that in these media the two carboxylate groups can be approached more easily than in the gas phase. The enzyme specifically reduces the activation free energy of the Claisen rearrangement about 3 kcal mol -1 more than that for the Cope reaction. This result is due to a larger transition state stabilization associated to the formation of a hydrogen bond between Arg90 and the ether oxygen. When this oxygen atom is changed by a methylene group, the interaction is lost and Arg90 moves inside the active site establishing stronger interactions with one of the carboxylate groups. This fact yields a more intense rearrangement of the substrate structure. Comparing two reactions in the same enzyme, we have been able to obtain conclusions about the relative magnitude of the substrate preorganization and transition state stabilization effects. Transition state stabilization seems to be the dominant effect in this case.

Journal of the American Chemical Society, 2008

Registro de acceso restringido Este recurso no está disponible en acceso abierto por política de ... more Registro de acceso restringido Este recurso no está disponible en acceso abierto por política de la editorial. No obstante, se puede acceder al texto completo desde la Universitat Jaume I o si el usuario cuenta con suscripción. Registre d'accés restringit Aquest recurs no està disponible en accés obert per política de l'editorial. No obstant això, es pot accedir al text complet des de la Universitat Jaume I o si l'usuari compta amb subscripció. Restricted access item This item isn't open access because of publisher's policy. The full--text version is only available from Jaume I University or if the user has a running suscription to the publisher's contents.

Journal of the American Chemical Society, 2013

Isotopic substitution ( 15 N, 13 C, 2 H) of a catalytically compromised variant of Escherichia co... more Isotopic substitution ( 15 N, 13 C, 2 H) of a catalytically compromised variant of Escherichia coli dihydrofolate reductase, EcDHFR-N23PP/S148A, has been used to investigate the effect of these mutations on catalysis. The reduction of the rate constant of the chemical step in the EcDHFR-N23PP/S148A catalyzed reaction is essentially a consequence of an increase of the quasi-classical free energy barrier and to a minor extent of an increased number of recrossing trajectories on the transition state dividing surface. Since the variant enzyme is less well set up to catalyze the reaction, a higher degree of active site reorganization is needed to reach the TS. Although millisecond active site motions are lost in the variant, there is greater flexibility on the femtosecond time scale. The "dynamic knockout" EcDHFR-N23PP/S148A is therefore a "dynamic knock-in" at the level of the chemical step, and the increased dynamic coupling to the chemical coordinate is in fact detrimental to catalysis. This finding is most likely applicable not just to hydrogen transfer in EcDHFR but also to other enzymatic systems.

Chemistry - A European Journal, 2003

In this paper a deeper insight into the chorismate-to prephenate-rearrangement, catalyzed by Baci... more In this paper a deeper insight into the chorismate-to prephenate-rearrangement, catalyzed by Bacillus subtilis chorismate mutase, is provided by means of a combination of statistical quantum mechanics/molecular mechanics simulation methods and hybrid potential energy surface exploration techniques. The main aim of this work is to present an estimation of the preorganization and reorganization terms of the enzyme catalytic rate enhancement. To analyze the first of these, we have studied different conformational equilibria of chorismate in aqueous solution and in the enzyme active site. Our conclusion is that chorismate mutase preferentially binds the reactive conformer of the substrate--that presenting a structure similar to the transition state of the reaction to be catalyzed--with shorter distances between the carbon atoms to be bonded and more diaxial character. With respect to the reorganization effect, an energy decomposition analysis of the potential energies of the reactive reactant and of the reaction transition state in aqueous solution and in the enzyme shows that the enzyme structure is better adapted to the transition structure. This means not only a more negative electrostatic interaction energy with the transition state but also a low enzyme deformation contribution to the energy barrier. Our calculations reveal that the structure of the enzyme is responsible for stabilizing the transition state structure of the reaction, with concomitant selection of the reactive form of the reactants. This is, the same enzymatic pattern that stabilizes the transition structure also promotes those reactant structures closer to the transition structure (i.e., the reactive reactants). In fact, both reorganization and preorganization effects have to be considered as the two faces of the same coin, having a common origin in the effect of the enzyme structure on the energy surface of the substrate.

Chemistry - A European Journal, 2008

Chemical Society Reviews, 2008

The purpose of this tutorial review is to illustrate the way to design new and powerful catalysts... more The purpose of this tutorial review is to illustrate the way to design new and powerful catalysts. The first possibility to get a biological catalyst for a given chemical process is to use existing enzymes that catalyze related reactions. The second possibility is the use of immune systems that recognize stable molecules resembling the transition structure of the target reaction. We finally show how computational techniques are able to provide an enormous quantity of information, providing clues to guide the development of new biological catalysts.

Biophysical Journal, 2008

is one of the three human immunodeficiency virus type 1 (HIV-1) enzymes essential for effective v... more is one of the three human immunodeficiency virus type 1 (HIV-1) enzymes essential for effective viral replication. Recently, mutation studies have been reported that have shown that a certain degree of viral resistance to diketo acids (DKAs) appears when some amino acid residues of the IN active site are mutated. Mutations represent a fascinating experimental challenge, and we invite theoretical simulations for the disclosure of still unexplored features of enzyme reactions. The aim of this work is to understand the molecular mechanisms of HIV-1 IN drug resistance, which will be useful for designing anti-HIV inhibitors with unique resistance profiles. In this study, we use molecular dynamics simulations, within the hybrid quantum mechanics/ molecular mechanics (QM/MM) approach, to determine the protein-ligand interaction energy for wild-type and N155S mutant HIV-1 IN, both complexed with a DKA. This hybrid methodology has the advantage of the inclusion of quantum effects such as ligand polarization upon binding, which can be very important when highly polarizable groups are embedded in anisotropic environments, for example in metal-containing active sites. Furthermore, an energy terms decomposition analysis was performed to determine contributions of individual residues to the enzyme-inhibitor interactions. The results reveal that there is a strong interaction between the Lys-159, Lys-156, and Asn-155 residues and Mg 21 cation and the DKA inhibitor. Our calculations show that the binding energy is higher in wild-type than in the N155S mutant, in accordance with the experimental results. The role of the mutated residue has thus been checked as maintaining the structure of the ternary complex formed by the protein, the Mg 21 cation, and the inhibitor. These results might be useful to design compounds with more interesting anti-HIV-1 IN activity on the basis of its three-dimensional structure.

Biochemistry, 2014

Because of the increasing resistance of malaria parasites to antimalarial drugs, the lack of high... more Because of the increasing resistance of malaria parasites to antimalarial drugs, the lack of highly effective vaccines, and an inadequate control of mosquito vectors, the problem is growing, especially in the developing world. New approaches to drug development are consequently required. One of the proteases involved in the degradation of human hemoglobin is named falcipain-2 (FP2), which has emerged as a promising target for the development of novel antimalarial drugs. However, very little is known about the inhibition of FP2. In this paper, the inhibition of FP2 by the epoxysuccinate E64 has been studied by molecular dynamics (MD) simulations using hybrid AM1d/MM and M06-2X/ MM potentials to obtain a complete picture of the possible free energy reaction paths. A thorough analysis of the reaction mechanism has been conducted to understand the inhibition of FP2 by E64. According to our results, the irreversible attack of Cys42 on E64 can take place on both carbon atoms of the epoxy ring because both processes present similar barriers. While the attack on the C2 atom presents a slightly smaller barrier (12.3 vs 13.6 kcal mol −1 ), the inhibitor−protein complex derived from the attack on C3 appears to be much more stabilized. In contrast to previous hypotheses, our results suggest that residues such as Gln171, Asp170, Gln36, Trp43, Asn81, and even His174 would be anchoring the inhibitor in a proper orientation for the reaction to take place. These results may be useful for the rational design of new compounds with higher inhibitory activity. . performed the bulk of computational simulations. S.M. wrote the codes and guided some of the calculations. S.M., S.F., and V.M. designed the research. K.A., S.F., and V.M. cowrote the first version of the manuscript. All the authors analyzed data, and V.M. wrote the final version of the manuscript.

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.

Theoretical Chemistry Accounts, 2011

Salicylate synthase from Mycobacterium tuberculosis, MbtI, initiates the biosynthesis of sideroph... more Salicylate synthase from Mycobacterium tuberculosis, MbtI, initiates the biosynthesis of siderophores by converting chorismate to salicylate. Nevertheless, three more distinct activities for wild-type MbtI have been detected in vitro: isochorismate synthase, isochorismate pyruvate lyase, and chorismate mutase. In this work, hybrid Quantum Mechanics/Molecular Mechanics methods have been used to get the first simulation of the chorismate mutase activity of MbtI. The results show how the reaction proceeds by means of a [3,3] sigmatropic rearrangement with free energy barrier in very good agreement with experiments.

Physical Chemistry Chemical Physics, 2012

Salicylate synthase from Mycobacterium tuberculosis, MbtI, is a highly promiscuous Mg 2+ dependen... more Salicylate synthase from Mycobacterium tuberculosis, MbtI, is a highly promiscuous Mg 2+ dependent enzyme with up to four distinct activities detected in vitro: isochorismate synthase (IS), isochorismate pyruvate lyase (IPL), salicylate synthase (SS) and chorismate mutase (CM). In this paper, Molecular Dynamic (MD) simulations employing hybrid quantum mechanics/molecular mechanics (QM/MM) potentials have been carried out to get a detailed knowledge of the IS and the IPL activities at the molecular level. According to our simulations, the architecture of the MbtI active site allows catalyzing the two reactions: the isochorismate formation, by means of a stepwise mechanism, and the salicylate production from isochorismate, that appears to be pericyclic in nature. Findings also explain the role of the magnesium cation and the pH dependence activity experimentally observed in MbtI. Mg 2+ would be polarizing and pre-organizing the substrate and active site, as well as shifting the pK a values of key active site residues. w Electronic supplementary information (ESI) available: to S7 containing X-ray structures of Irp9 (2FN1) and MbtI (2G5F), 2D-PMFs of reaction corresponding to the IS activity of MbtI corrected at the M06-2X level and representative structures of the substrate in chorismate, intermediate, isochorismate and transition states located in the active site of the MbtI, 2D-PMF of reaction corresponding to the IPL activity of MbtI corrected at the M06-2X level and representative structures of the substrate in the isochorismate, transition state and products (salicylate and pyruvate), representative snapshots of the K205Q mutated enzyme compared to the wild-type and time dependent RMSD during equilibration of the system. See

Journal of computational chemistry, Jan 19, 2015

CYP19A1 aromatase is a member of the Cytochrome P450 family of hemeproteins, and is the enzyme re... more CYP19A1 aromatase is a member of the Cytochrome P450 family of hemeproteins, and is the enzyme responsible for the final step of the androgens conversion into the corresponding estrogens, via a three-step oxidative process. For this reason, the inhibition of this enzyme plays an important role in the treatment of hormone-dependent breast cancer. The first catalytic subcycle, corresponding to the hydroxilation of androstenedione, has been proposed to occur through a first hydrogen abstraction and a subsequent oxygen rebound step. In present work, we have studied the mechanism of the first catalytic subcycle by means of hybrid quantum mechanics/molecular mechanics methods. The inclusion of the protein flexibility has been achieved by means of Free Energy Perturbation techniques, giving rise to a free energy of activation for the hydrogen abstraction step of 13.5 kcal/mol. The subsequent oxygen rebound step, characterized by a small free energy barrier (1.5 kcal/mol), leads to the hydr...

ACS catalysis, Jan 7, 2015

In the last decade L-Lactate Dehydrogenase (LDH) has become an extremely useful marker in both cl... more In the last decade L-Lactate Dehydrogenase (LDH) has become an extremely useful marker in both clinical diagnosis and in monitoring the course of many human diseases. It has been assumed from the 80s that the full catalytic process of LDH starts with the binding of the cofactor and the substrate followed by the enclosure of the active site by a mobile loop of the protein before the reaction to take place. In this paper we show that the chemical step of the LDH catalyzed reaction can proceed within the open loop conformation, and the different reactivity of the different protein conformations would be in agreement with the broad range of rate constants measured in single molecule spectrometry studies. Starting from a recently solved X-ray diffraction structure that presented an open loop conformation in two of the four chains of the tetramer, QM/MM free energy surfaces have been obtained at different levels of theory. Depending on the level of theory used to describe the electronic s...

Journal of Physical Chemistry B, 2008

We discuss the role of the protein in controlling the absorption spectra of photoactive yellow pr... more We discuss the role of the protein in controlling the absorption spectra of photoactive yellow protein (PYP), the archetype xanthopsin photoreceptor, using quantum mechanics/molecular mechanics (QM/MM) methods based on ab initio multireference perturbation theory, combined with molecular dynamics (MD) simulations. It is shown that in order to get results in agreement with the experimental data, it is necessary to use a model that allows for a proper relaxation of the whole system and treats the states involved in the electronic spectrum in a balanced way, avoiding biased results due to the effect of nonrepresentative electrostatic interactions on the chromophore.

The journal of physical chemistry. A, Jan 19, 2006

Catalytic antibodies are very interesting not only because of the rate enhancement of the reactio... more Catalytic antibodies are very interesting not only because of the rate enhancement of the reactions that they catalyze but also because of the selectivities they can achieve that are sometimes not present in natural enzyme processes. We have selected the study of the stereoselectivity of the matured AZ28 that catalyzes an oxy-Cope rearrangement. For this particular case, the presence of a chiral center in the substrate provokes the existence of two different enantiomers, R and S. Furthermore, it is also possible to locate two different orientations for the hydroxyl group in the central ring of the substrate in the transition state, equatorial and axial, rendering two different conformers. In this paper we present the free energy profiles obtained for different substrate isomers in the cavity created by the matured catalytic antibody. Our simulations have reproduced the stereoselectivity of the matured AZ28, differentiating between the axial or equatorial orientations and preferentia...

The Journal of Physical Chemistry B, 2011

Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations were performed to ... more Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations were performed to investigate the methylation of 6-mercaptopurine catalyzed by thiopurine S-methyltransferase. Several setups with different tautomeric forms and orientations of the substrate were considered. It is found that, with the orientation in chain A of the X-ray structure, the substrate can form an ideal near-attack configuration for the methylation reaction, which may take place after the deprotonation of the substrate by the conserved residue Asp23 through a water chain. The potential of mean force (PMF) of the methyl-transfer step for the most favorable pathway is 19.6 kcal/ mol, which is in good agreement with the available experimental rate constant data.

Organic & Biomolecular Chemistry, 2003

Kinetic isotope effects have been computed for the Claisen rearrangement of chorismate to prephen... more Kinetic isotope effects have been computed for the Claisen rearrangement of chorismate to prephenate in aqueous solution and in the active site of chorismate mutase from B. subtilus. These included primary 13C and 18O and secondary 3H effects for substitutions at the bond-making and bond-breaking positions. The initial structures of the putative stationary points on the potential energy surface, required for the calculations of isotope effects using the CAMVIB/CAMISO programs, have been selected from hybrid QM/MM molecular dynamical simulations using the DYNAMO program. Refinement of the reactant complex and transition-state structures has been carried out by means of AM1/CHARMM24/TIP3P calculations using the GRACE program, with full gradient relaxation of the position of > 5200 atoms for the enzymic simulations, and with a box containing 711 water molecules for the corresponding reaction in aqueous solution. Comparison of these results, and of gas phase calculations, with experimental data has shown that the chemical rearrangement is largely rate-determining for the enzyme mechanism. Inclusion of the chorismate conformational pre-equilibrium step in the modelled kinetic scheme leads to better agreement between recent experimental data and theoretical predictions. These results provide new information on an important enzymatic transformation, and the key factors responsible for the kinetics of its molecular mechanism are clarified. Treatment of the enzyme and/or solvent environment by means of a large and flexible model is absolutely essential for prediction of kinetic isotope effects.

Nature Chemistry, 2013

Conformational changes are known to be able to drive an enzyme through its catalytic cycle, allow... more Conformational changes are known to be able to drive an enzyme through its catalytic cycle, allowing, for example, substrate binding or product release. However, the influence of protein motions on the chemical step is a controversial issue. One proposal is that the simple equilibrium fluctuations incorporated into transition-state theory are insufficient to account for the catalytic effect of enzymes and that protein motions should be treated dynamically. Here, we propose the use of free-energy surfaces, obtained as a function of both a chemical coordinate and an environmental coordinate, as an efficient way to elucidate the role of protein structure and motions during the reaction. We show that the structure of the protein provides an adequate environment for the progress of the reaction, although a certain degree of flexibility is needed to attain the full catalytic effect. However, these motions do not introduce significant dynamical corrections to the rate constant and can be described as equilibrium fluctuations.

Journal of the American Chemical Society, 2009

The isochorismate pyruvate lyase (IPL) from Pseudomonas aeruginosa, designated as PchB, catalyzes... more The isochorismate pyruvate lyase (IPL) from Pseudomonas aeruginosa, designated as PchB, catalyzes the transformation of isochorismate into pyruvate and salicylate, but it also catalyzes the rearrangement of chorismate into prephenate, suggesting that both reactions may proceed by a pericyclic mechanism. In this work, molecular dynamics simulations employing hybrid quantum mechanics/molecular mechanics methods have been carried out to get a detailed knowledge of the reaction mechanism of PchB. The results provide a theoretical rate constant enhancement by comparison with the reaction in solution, in agreement with the experimental data, and confirm the pericyclic nature of the reaction mechanism. The robustness of this promiscuous enzyme has been checked by considering the impact of Ala37Ile mutation, previously proposed by us to improve the secondary chorismate mutase (CM) activity. The effect of this mutation, which was shown to increase the rate constant for the CM activity by a factor of 10(3), also increases the IPL catalytic efficiency, although only by a factor of 6.

Journal of the American Chemical Society, 2006

In some enzymatic systems large conformational changes are coupled to the chemical step, in such ... more In some enzymatic systems large conformational changes are coupled to the chemical step, in such a way that dispersion of rate constants can be observed in single-molecule experiments, each corresponding to the reaction from a different reactant valley. Under this perspective here we present a computational study of pyruvate to lactate transformation catalyzed by lactate dehydrogenase. The reaction consists of a hydride transfer and a proton transfer that seem to take place concertedly. The degree of asynchronicity and the energy barrier depend on the particular starting reactant valley. In order to estimate rate constants we used a free energy perturbation technique adapted to follow the intrinsic reaction coordinate for several possible reaction paths. Tunneling effects are also obtained with a slightly modified version of the ensemble-averaged variational transition state theory with multidimensional tunneling contributions. According to our results the closure of the active site by means of a flexible loop can lead to the formation of different reactant complexes displaying different features in the disposition of some key residues (such as Arg109), interactions with the substrate and number of water molecules in the active site. The chemical step of the reaction takes place with a different reaction rate from each of these complexes. Finally, primary kinetic isotope effects for replacement of the transferring hydrogen of the cofactor with a deuteride are in good agreement with experimental observations, thus validating our methodology.

Journal of the American Chemical Society, 2004

In this work we present a detailed analysis of the activation free energies and averaged interact... more In this work we present a detailed analysis of the activation free energies and averaged interactions for the Claisen and Cope rearrangements of chorismate and carbachorismate catalyzed by Bacillus subtilis chorismate mutase (BsCM) using quantum mechanics/molecular mechanics (QM/MM) simulation methods. In gas phase, both reactions are described as concerted processes, with the activation free energy for carbachorismate being about 10-15 kcal mol -1 larger than for chorismate, at the AM1 and B3LYP/6-31G* levels. Aqueous solution and BsCM active site environments reduce the free energy barriers for both reactions, due to the fact that in these media the two carboxylate groups can be approached more easily than in the gas phase. The enzyme specifically reduces the activation free energy of the Claisen rearrangement about 3 kcal mol -1 more than that for the Cope reaction. This result is due to a larger transition state stabilization associated to the formation of a hydrogen bond between Arg90 and the ether oxygen. When this oxygen atom is changed by a methylene group, the interaction is lost and Arg90 moves inside the active site establishing stronger interactions with one of the carboxylate groups. This fact yields a more intense rearrangement of the substrate structure. Comparing two reactions in the same enzyme, we have been able to obtain conclusions about the relative magnitude of the substrate preorganization and transition state stabilization effects. Transition state stabilization seems to be the dominant effect in this case.

Journal of the American Chemical Society, 2008

Registro de acceso restringido Este recurso no está disponible en acceso abierto por política de ... more Registro de acceso restringido Este recurso no está disponible en acceso abierto por política de la editorial. No obstante, se puede acceder al texto completo desde la Universitat Jaume I o si el usuario cuenta con suscripción. Registre d'accés restringit Aquest recurs no està disponible en accés obert per política de l'editorial. No obstant això, es pot accedir al text complet des de la Universitat Jaume I o si l'usuari compta amb subscripció. Restricted access item This item isn't open access because of publisher's policy. The full--text version is only available from Jaume I University or if the user has a running suscription to the publisher's contents.

Journal of the American Chemical Society, 2013

Isotopic substitution ( 15 N, 13 C, 2 H) of a catalytically compromised variant of Escherichia co... more Isotopic substitution ( 15 N, 13 C, 2 H) of a catalytically compromised variant of Escherichia coli dihydrofolate reductase, EcDHFR-N23PP/S148A, has been used to investigate the effect of these mutations on catalysis. The reduction of the rate constant of the chemical step in the EcDHFR-N23PP/S148A catalyzed reaction is essentially a consequence of an increase of the quasi-classical free energy barrier and to a minor extent of an increased number of recrossing trajectories on the transition state dividing surface. Since the variant enzyme is less well set up to catalyze the reaction, a higher degree of active site reorganization is needed to reach the TS. Although millisecond active site motions are lost in the variant, there is greater flexibility on the femtosecond time scale. The "dynamic knockout" EcDHFR-N23PP/S148A is therefore a "dynamic knock-in" at the level of the chemical step, and the increased dynamic coupling to the chemical coordinate is in fact detrimental to catalysis. This finding is most likely applicable not just to hydrogen transfer in EcDHFR but also to other enzymatic systems.

Chemistry - A European Journal, 2003

In this paper a deeper insight into the chorismate-to prephenate-rearrangement, catalyzed by Baci... more In this paper a deeper insight into the chorismate-to prephenate-rearrangement, catalyzed by Bacillus subtilis chorismate mutase, is provided by means of a combination of statistical quantum mechanics/molecular mechanics simulation methods and hybrid potential energy surface exploration techniques. The main aim of this work is to present an estimation of the preorganization and reorganization terms of the enzyme catalytic rate enhancement. To analyze the first of these, we have studied different conformational equilibria of chorismate in aqueous solution and in the enzyme active site. Our conclusion is that chorismate mutase preferentially binds the reactive conformer of the substrate--that presenting a structure similar to the transition state of the reaction to be catalyzed--with shorter distances between the carbon atoms to be bonded and more diaxial character. With respect to the reorganization effect, an energy decomposition analysis of the potential energies of the reactive reactant and of the reaction transition state in aqueous solution and in the enzyme shows that the enzyme structure is better adapted to the transition structure. This means not only a more negative electrostatic interaction energy with the transition state but also a low enzyme deformation contribution to the energy barrier. Our calculations reveal that the structure of the enzyme is responsible for stabilizing the transition state structure of the reaction, with concomitant selection of the reactive form of the reactants. This is, the same enzymatic pattern that stabilizes the transition structure also promotes those reactant structures closer to the transition structure (i.e., the reactive reactants). In fact, both reorganization and preorganization effects have to be considered as the two faces of the same coin, having a common origin in the effect of the enzyme structure on the energy surface of the substrate.

Chemistry - A European Journal, 2008

Chemical Society Reviews, 2008

The purpose of this tutorial review is to illustrate the way to design new and powerful catalysts... more The purpose of this tutorial review is to illustrate the way to design new and powerful catalysts. The first possibility to get a biological catalyst for a given chemical process is to use existing enzymes that catalyze related reactions. The second possibility is the use of immune systems that recognize stable molecules resembling the transition structure of the target reaction. We finally show how computational techniques are able to provide an enormous quantity of information, providing clues to guide the development of new biological catalysts.

Biophysical Journal, 2008

is one of the three human immunodeficiency virus type 1 (HIV-1) enzymes essential for effective v... more is one of the three human immunodeficiency virus type 1 (HIV-1) enzymes essential for effective viral replication. Recently, mutation studies have been reported that have shown that a certain degree of viral resistance to diketo acids (DKAs) appears when some amino acid residues of the IN active site are mutated. Mutations represent a fascinating experimental challenge, and we invite theoretical simulations for the disclosure of still unexplored features of enzyme reactions. The aim of this work is to understand the molecular mechanisms of HIV-1 IN drug resistance, which will be useful for designing anti-HIV inhibitors with unique resistance profiles. In this study, we use molecular dynamics simulations, within the hybrid quantum mechanics/ molecular mechanics (QM/MM) approach, to determine the protein-ligand interaction energy for wild-type and N155S mutant HIV-1 IN, both complexed with a DKA. This hybrid methodology has the advantage of the inclusion of quantum effects such as ligand polarization upon binding, which can be very important when highly polarizable groups are embedded in anisotropic environments, for example in metal-containing active sites. Furthermore, an energy terms decomposition analysis was performed to determine contributions of individual residues to the enzyme-inhibitor interactions. The results reveal that there is a strong interaction between the Lys-159, Lys-156, and Asn-155 residues and Mg 21 cation and the DKA inhibitor. Our calculations show that the binding energy is higher in wild-type than in the N155S mutant, in accordance with the experimental results. The role of the mutated residue has thus been checked as maintaining the structure of the ternary complex formed by the protein, the Mg 21 cation, and the inhibitor. These results might be useful to design compounds with more interesting anti-HIV-1 IN activity on the basis of its three-dimensional structure.