Research

Many fundamental biological processes, such as enzyme catalysis and covalent ligand-protein interactions, involve mechanisms in which chemical bonds are formed and/or broken, thus involving significant electronic changes. For instance, when oxygen binds to the heme iron atom of hemoglobin and other heme enzymes, the metal coordination and its spin state change from a penta-coordinated iron in a quintuplet state to an hexa-coordinated iron whose electron distribution has long been debated. In the context of carbohydrate-active enzymes, suitably designed inhibitors can react with catalytic residues in the active site glycosidases and can be converted into activity-based probes, allowing to investigate enzyme identity and function.   

Our research is focused on the study of reactive biological processes at atomic and electronic detail, using computer simulation. Our main tools are ab initio molecular dynamics (e.g. Car-Parrinello MD), enhanced sampling methods (e.g. metadynamics and path-CV methods), classical MD and hybrid quantum mechanics/molecular mechanics (QM/MM).  Most projects are being performed in collaboration with experimental groups of synthetic, chemical and structural biology. 

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Current research interests:

Enzymatic synthesis and degradation of carbohydrates

Understanding the main factors governing the interaction of an enzyme with its substrate is a topic of ongoing interest in molecular and computational biology. It is now widely recognized that enzymes slightly change shape as the substrate binds (Koshland, 1958). In some cases (e.g. glycosyl hydrolases), enzyme-substrate interactions affect the structure of the substrate as well. Computer simulation represent a powerful aid to the identification of the precise substrate conformation, since it allows to investigate directly the native substrate-bound enzyme. We are interested in understanding the changes induced by the enzyme and/or carbohydrate upon binding and during the enzymatic reaction in two of the main groups of carbohydrate-active enzymes (CAZymes): glycoside hydrolases (GHs) and glycosyltransferases (GTs). Our research is also intended to help in the design of inhibitors for CAZymes involved in disease.

GHs and GTs are highly specific enzymes responsible of the hydrolysis and formation of glycosidic bonds in carbohydrates, respectively. The knowledge of their enzymatic mechanism at the molecular level is crucial to understand how carbohydrates are processed in the organisms. In 2006 we investigated the dynamics of the carbohydrate-protein interaction in Bacillus 1-3,1-4-beta-glucanase as a test case. Our study confirmed that one sugar ring of the substrate undergoes a distortion (from a chair to a skew-boat conformation) upon binding to the protein and that this distortion favors the enzymatic reaction (JBC 2006, 282, 1432-1441). By means of ab initio QM/MM metadynamics simulations we also showed that the distortions occurring in beta-glucoside hydrolases can be rationalized from the conformational free energy landscape of one sugar unit (JACS 2007, 129, 10686-10693), which has been extended to a number of GHs (reviewed in Acc. Chem. Res. 2012, 45, 308-312, JACS 2015, 137, 7528−7547, Curr. Opin. Chem. Biol. 219, 53, 183-191 and Curr. Opin. Struct. Biol. 2020, 62, 79-92).

During the last years we have investigated the molecular mechanisms of glycosidic bond formation in Leloir-type retaining GTs, capturing the short-lived oxocarbenium ion intermediate that is formed during the enzymatic reaction (Angew. Chem. Int. Ed. 2011, 50, 10897-10901), which is elusive to most experimental probes. This type of reaction, named as "front-face" or "SNi-like" in the literature, is nowadays the most accepted reaction mechanism for retaining GTs. We have demonstrated that it is also operative for O-glycosylation catalyzed by by polypeptide N-Acetylgalactosaminyltransferase 2 (Angew. Chem. Int. Ed. 2014, 53, 8206–8210) and for the initial stages of glycogen biosynthesis (Nature 2018, 563, 235-240).

Our next goal is to predict specific modifications of these enzymes that could affect its catalytic activity, as well as designing experiments to test these predictions. These investigation is performed in collaboration with several experimental groups of biochemistry, synthetic chemistry, structural and chemical biology (A. Planas, IQS-Universitat Ramon Llull, Barcelona;  G. J. Davies, University of York, UK; S. Williams, University of Melbourne, Australia; R. Hurtado-Guerrero, Universidad de Zaragoza, Spain, B. G. Davis, U. Oxford; H. S. Overkleeft, Leiden University, NL).

Catalase and peroxidase catalytic processes

Heme peroxidases are an important group of enzymes that are widespread in nature and found in plants, fungi, bacteria and mammals. These enzymes use hydrogen peroxide to oxidize a variety of organic substrates (each enzyme oxidizes a different substrate). They calalyze the reaction: 2Ared  + H2O2 → 2Aox  + 2H2O, where Ared is the organic substrate (an electron donor) and Aox dot is the oxidized substrate. The substrate is usually an organic molecule (e.g. carboxilic acids and aromatic phenols). Heme catalases (also named as catalatic hydroperoxidases) are similar enzymes, whose main function is the degradation of hydrogen peroxide to oxygen and water (2H2O2 → 2H2O + O2). They are also present in all forms of life. Both peroxidases and catalases are hemeproteins, i.e. have a heme active center (right figure) and share many mechanistic aspects such as the formation of the so-called compound I intermediate (Cpd I), which is often an oxoferryl porphyrin cation radical (see JACS 2007, 129, 6346-6347, collaboration with S. Shaik). Catalase-peroxidases (KatGs) are bifunctional enzymes, with both catalase and peroxidase activities, that are responsible for the activation of isoniazid as an anti-tubercular drug in Mycobacterium tuberculosis. Both the catalytic mechanism of the enzyme and the mechanism of drug activation are unknown, although several hypotheses have been put forward. One of the major questions is how the switch between the catalase and peroxidase activities takes place (i.e. the electronic/structural basis of the enzyme bifunctionality). In collaboration with Profs. I. Fita (IBMB-CSIC, Barcelona) and P. Loewen (University of Manitoba, Canada) we are investigating  the catalytic cycle of KatGs (see JACS 2014, 136, 7249−7252 and Chem. Eur. J. 2018, 24, 5388-5395), as well as the binding of the isoniazid drug (J. Phys. Chem. B 2014, 118, 2924−2931). We are also interested in uncovering the mechanisms of monofunctional catalases and peroxidases (see JACS 2009, 131, 11751–11761 and JACS 137, 11170−11178).

Projectes de màster (TFM) i treball fi de grau (TFG) (only available in Catalan)