Carme Rovira

The Molecular Mechanism of the Catalase Reaction

Catalases are ubiquitous enzymes that prevent cell oxidative damage by degrading hydrogen peroxide to water and oxygen (2H(2)O(2) --> 2 H(2)O + O(2)) with high efficiency. The enzyme is first oxidized to a high-valent iron intermediate, known as Compound I (Cpd I) which, in contrast to other hydroperoxidases, is reduced back to the resting state by further reacting with H(2)O(2). By means of hybrid QM/MM Car-Parrinello metadynamics simulations, we have investigated the mechanism of the reduction of Compound I by H(2)O(2) in Helicobacter pylori catalase (HPC) and Penicillium vitale catalase (PVC). We found that the Cpd I-H(2)O(2) complex evolves to a Cpd II-like species through the transfer of a hydrogen atom from the peroxide to the oxoferryl unit. To complete the reaction, two mechanisms may be operative: a His-mediated (Fita-Rossmann) mechanism, which involves the distal His as an acid-base catalyst mediating the transfer of a proton (associated with an electron transfer), and a direct mechanism, in which a hydrogen atom transfer occurs. Independently of the mechanism, the reaction proceeds by two one-electron transfers rather than one two-electron transfer, as has long been the lore. The calculations provide a detailed view of the atomic and electronic reorganizations during the reaction, and highlight the key role of the distal residues to assist the reaction. Additional calculations on the in silico HPC His56Gly mutant and gas-phase models provide clues to understand the requirements for the reaction to proceed with low barriers.

Structural basis for the high all-trans-retinaldehyde reductase activity of the tumor marker AKR1B10

AKR1B10 is a human aldo-keto reductase (AKR) found to be elevated in several cancer types and in precancerous lesions. In vitro, AKR1B10 exhibits a much higher retinaldehyde reductase activity than any other human AKR, including AKR1B1 (aldose reductase). We here demonstrate that AKR1B10 also acts as a retinaldehyde reductase in vivo. This activity may be relevant in controlling the first step of retinoic acid synthesis. Up-regulation of AKR1B10, resulting in retinoic acid depletion, may lead to cellular proliferation. Both in vitro and in vivo activities of AKR1B10 were inhibited by tolrestat, an AKR1B1 inhibitor developed for diabetes treatment. The crystal structure of the ternary complex AKR1B10–NADP+–tolrestat was determined at 1.25-Å resolution. Molecular dynamics models of AKR1B10 and AKR1B1 with retinaldehyde isomers and site-directed mutagenesis show that subtle differences at the entrance of the retinoid-binding site, especially at position 125, are determinant for the all-trans-retinaldehyde specificity of AKR1B10. Substitutions in the retinaldehyde cyclohexene ring also influence the specificity. These structural features should facilitate the design of specific inhibitors, with potential use in cancer and diabetes treatments.

Influence of the heme pocket conformation on the structure and vibrations of the Fe-CO bond in myoglobin: a QM/MM density functional study.

The influence of the distal pocket conformation on the structure and vibrations of the heme-CO bond in carbonmonoxy myoglobin (MbCO) is investigated by means of hybrid QM/MM calculations based on density functional theory combined with a classical force field. It is shown that the heme-CO structure (QM treated) is quite rigid and not influenced by the distal pocket conformation (MM treated). This excludes any relation between FeCO distortions and the different CO absorptions observed in the infrared spectra of MbCO (A states). In contrast, both the CO stretch frequency and the strength of the CO...His64 interaction are very dependent on the orientation and tautomerization state of His64. Our calculations indicate that the CO...N(epsilon) type of approach does not contribute to the A states, whereas the CO...H-N(epsilon) interaction is the origin of the A(1) and A(3) states, the His64 residue being protonated at N(epsilon). The strength of the CO...His64 interaction is quantified, in comparison with the analogous O(2)...His64 interaction and with the observed changes in the CO stretch frequency. Additional aspects of the CO...His64 interaction and its biological implications are discussed.

Privateer: software for the conformational validation of carbohydrate structures.

833 from the start and may require rebuilding (Fig. 1a). At lower resolutions, higher-energy conformations may appear as a consequence of underparameterized refinement, in spite of starting from a correct input model. These can be corrected during both realand reciprocalspace refinement (Fig. 1b), provided that enough restraints are introduced to balance the parameter-to-observation ratio. It is such examples that emphasize the problems of modeling sugars where the density is poor. Both published structures have high-energy conformations that cannot be deduced from the electron density, although for different reasons. In the first case, holds the conformation as determined by the Cremer-Pople algorithm4, as well as stereochemical and geometric information, for use as a reference upon validation. Additionally, puckering amplitudes4 of pyranoses are compared to those registered during ab initio metadynamics simulations of the conformational free-energy landscape of cyclohexane5. Privateer is able to feed results into Coot6 through its Python or Scheme scripting interface, loading models and maps automatically and flagging issues visually. Most of the conformational outliers detected at higher resolutions (<1.6 Å) are typically wrongly modeled Privateer (http://www.ccp4.ac.uk/html/ privateer.html) is a new software package aimed at the detection and prevention of conformational, regiochemical and stereochemical anomalies in cyclic monosaccharide structures. Carbohydrates, including Oand N-glycans attached to protein and lipid structures, are increasingly being studied in cellular biology. Crystallographic refinement of sugars is, however, poorly performed, thus leading to thousands of incorrect structures having been deposited in the Protein Data Bank (PDB)1,2. Although nomenclature validation has become possible in the past decade, with the introduction of tools such as PDB carbohydrate residue check (pdb-care)3, inappropriate refinement protocols at resolutions lower than 1.6 Å can still force a correct sugar into a highly improbable ring conformation, let alone distort one with chemical errors1. High-energy conformations are very infrequent in nature—perhaps even out of the question in N-glycans—and must always be backed by clear electron density. Otherwise, such conformations should be treated as outliers. Privateer identifies incorrect regiochemistry and stereochemistry and unlikely conformations. A real-space correlation coefficient against omit mFo – DFc electron density is also calculated as a quality-of-fit indicator. Bias-minimized map coefficients are exported automatically and can be subsequently used to assess identification of the sugars. Using this information, Privateer produces a visual checklist for rapid correction of the errors in real space and ensures that conformational preferences are accounted for during subsequent rebuilding and refinement. The software holds a manually curated database of supported monosaccharides based on the PDB Chemical Component Dictionary, whose entries contain coordinates for an energyminimized conformer that the PDB calculates upon new ligand depositions, by using Corina (Molecular Networks) or Omega (OpenEye). For each of these supported sugars, the database Privateer: software for the conformational validation of carbohydrate structures

Substrate Distortion in the Michaelis Complex of Bacillus 1,3–1,4-β-Glucanase INSIGHT FROM FIRST PRINCIPLES MOLECULAR DYNAMICS SIMULATIONS

The structure and dynamics of the enzyme-substrate complex of Bacillus 1,3–1,4-β-glucanase, one of the most active glycoside hydrolases, is investigated by means of Car-Parrinello molecular dynamics simulations (CPMD) combined with force field molecular dynamics (QM/MM CPMD). It is found that the substrate sugar ring located at the –1 subsite adopts a distorted 1S3 skew-boat conformation upon binding to the enzyme. With respect to the undistorted 4C1 chair conformation, the 1S3 skew-boat conformation is characterized by: (a) an increase of charge at the anomeric carbon (C1), (b) an increase of the distance between C1 and the leaving group, and (c) a decrease of the intraring O5-C1 distance. Therefore, our results clearly show that the distorted conformation resembles both structurally and electronically the transition state of the reaction in which the substrate acquires oxocarbenium ion character, and the glycosidic bond is partially broken. Together with analysis of the substrate conformational dynamics, it is concluded that the main determinants of substrate distortion have a structural origin. To fit into the binding pocket, it is necessary that the aglycon leaving group is oriented toward the β region, and the skew-boat conformation naturally fulfills this premise. Only when the aglycon is removed from the calculation the substrate recovers the all-chair conformation, in agreement with the recent determination of the enzyme product structure. The QM/MM protocol developed here is able to predict the conformational distortion of substrate binding in glycoside hydrolases because it accounts for polarization and charge reorganization at the –1 sugar ring. It thus provides a powerful tool to model E·S complexes for which experimental information is not yet available.

Aldo-keto reductases from the AKR1B subfamily: retinoid specificity and control of cellular retinoic acid levels.

NADP(H)-dependent cytosolic aldo-keto reductases (AKRs) have been added to the group of enzymes which contribute to oxidoreductive conversions of retinoids. Recently, we found that two members from the AKR1B subfamily (AKR1B1 and AKRB10) were active in the reduction of all-trans- and 9-cis-retinaldehyde, with K(m) values in the micromolar range, but with very different k(cat) values. With all-trans-retinaldehyde, AKR1B10 shows a much higher k(cat) value than AKR1B1 (18 min(-1)vs. 0.37 min(-1)) and a catalytic efficiency comparable to that of the best retinaldehyde reductases. Structural, molecular dynamics and site-directed mutagenesis studies on AKR1B1 and AKR1B10 point that subtle differences at the entrance of their retinoid-binding site, especially at position 125, are determinant for the all-trans-retinaldehyde specificity of AKR1B10. Substitutions in the retinoid cyclohexene ring, analyzed here further, also influence such specificity. Overall it is suggested that the rate-limiting step in the reaction mechanism with retinaldehyde differs between AKR1B1 and AKR1B10. In addition, we demonstrate here that enzymatic activity of AKR1B1 and AKR1B10 lowers all-trans- and 9-cis-retinoic acid-dependent trans-activation in living cells, indicating that both enzymes may contribute to pre-receptor regulation of retinoic acid and retinoid X nuclear receptors. This result supports that overexpression of AKR1B10 in cancer (an updated review on this topic is included) may contribute to dedifferentiation and tumor development.

Reaction Mechanisms in Carbohydrate-Active Enzymes: Glycoside Hydrolases and Glycosyltransferases. Insights from ab Initio Quantum Mechanics/Molecular Mechanics Dynamic Simulations

Carbohydrate-active enzymes such as glycoside hydrolases (GHs) and glycosyltransferases (GTs) are of growing importance as drug targets. The development of efficient competitive inhibitors and chaperones to treat diseases related to these enzymes requires a detailed knowledge of their mechanisms of action. In recent years, sophisticated first-principles modeling approaches have significantly advanced in our understanding of the catalytic mechanisms of GHs and GTs, not only the molecular details of chemical reactions but also the significant implications that just the conformational dynamics of a sugar ring can have on these mechanisms. Here we provide an overview of the progress that has been made in the past decade, combining molecular dynamics simulations with density functional theory to solve these sweet mysteries of nature.

Catalytic Itinerary in 1,3-1,4-β-Glucanase Unraveled by QM/MM Metadynamics. Charge Is Not Yet Fully Developed at the Oxocarbenium Ion-like Transition State

Retaining glycoside hydrolases (GHs), key enzymes in the metabolism of polysaccharides and glycoconjugates and common biocatalysts used in chemoenzymatic oligosaccharide synthesis, operate via a double-displacement mechanism with the formation of a glycosyl-enzyme intermediate. However, the degree of oxocarbenium ion character of the reaction transition state and the precise conformational itinerary of the substrate during the reaction, pivotal in the design of efficient inhibitors, remain elusive for many GHs. By means of QM/MM metadynamics, we unravel the catalytic itinerary of 1,3-1,4-β-glucanase, one of the most active GHs, belonging to family 16. We show that, in the Michaelis complex, the enzyme environment restricts the conformational motion of the substrate to stabilize a (1,4)B/(1)S(3) conformation of the saccharide ring at the -1 subsite, confirming that this distortion preactivates the substrate for catalysis. The metadynamics simulation of the enzymatic reaction captures the complete conformational itinerary of the substrate during the glycosylation reaction ((1,4)B/(1)S(3) -(4)E/(4)H(3) - (4)C(1)) and shows that the transition state is not the point of maximum charge development at the anomeric carbon. The overall catalytic mechanism is of dissociative type, and proton transfer to the glycosidic oxygen is a late event, clarifying previous kinetic studies of this enzyme.

Modulation of Aβ42 fibrillogenesis by glycosaminoglycan structure

The role of amyloid β (Aβ) peptide in the onset and progression of Alzheimers disease is linked to the presence of soluble Aβ species. Sulfated glycosaminoglycans (GAGs) promote Aβ fibrillogenesis and reduce the toxicity of the peptide in neuronal cell cultures, but a satisfactory rationale to explain these effects at the molecular level has not been provided yet. We have used circular dichroism, Fourier transform infrared spectroscopy, fluorescence microscopy and spectroscopy, protease digestion, atomic force microscopy (AFM), and molecular dynamics simulations to characterize the association of the 42‐residue fragment Aβ42 with sulfated GAGs, hyaluronan, chitosan, and poly(vinyl sulfate) (PVS). Our results indicate that the formation of stable Aβ42 fibrils is promoted by polymeric GAGs with negative charges placed in‐frame with the 4.8‐Å separating Aβ42 monomers within protofibrillar β‐sheets. Incubation of Aβ42 with excess sulfated GAGs and hyaluronan increased amyloid fibril content and resistance to proteolysis 2‐ to 5‐fold, whereas in the presence of the cationic polysaccharide chitosan, Aβ42 fibrillar species were reduced by 25% and sensitivity to protease degradation increased ∼3‐fold. Fibrils of intermediate stability were obtained in the presence of PVS, an anionic polymer with more tightly packed charges than GAGs. Important structural differences between Aβ42 fibrils induced by PVS and Aβ42 fibrils obtained in the presence of GAGs and hyaluronan were observed by AFM, whereas mainly precursor protofibrillar forms were detected after incubation with chitosan. Computed binding energies per peptide from −11.2 to −13.5 kcal/mol were calculated for GAGs and PVS, whereas a significantly lower value of −7.4 kcal/mol was obtained for chitosan. Taken together, our data suggest a simple and straightforward mechanism to explain the role of GAGs as enhancers of the formation of insoluble Aβ42 fibrils trapping soluble toxic forms.—Valle‐Delgado, J. J., Alfonso‐Prieto, M., de Groot, N. S., Ventura, S., Samitier, J., Rovira, C., Fernàndez‐Busquets, X. Modulation of Aβ42 fibrillogenesis by glycosaminoglycan structure. FASEB J. 24, 4250–4261 (2010). www.fasebj.org

Essential role of proximal histidine-asparagine interaction in mammalian peroxidases

In heme enzymes belonging to the peroxidase-cyclooxygenase superfamily the proximal histidine is in close interaction with a fully conserved asparagine. The crystal structure of a mixture of glycoforms of myeloperoxidase (MPO) purified from granules of human leukocytes prompted us to revise the orientation of this asparagine and the protonation status of the proximal histidine. The data we present contrast with previous MPO structures, but are strongly supported by molecular dynamics simulations. Moreover, comprehensive analysis of published lactoperoxidase structures suggest that the described proximal heme architecture is a general structural feature of animal heme peroxidases. Its importance is underlined by the fact that the MPO variant N421D, recombinantly expressed in mammalian cell lines, exhibited modified spectral properties and diminished catalytic activity compared with wild-type recombinant MPO. It completely lost its ability to oxidize chloride to hypochlorous acid, which is a characteristic feature of MPO and essential for its role in host defense. The presented crystal structure of MPO revealed further important differences compared with the published structures including the extent of glycosylation, interaction between light and heavy polypeptides, as well as heme to protein covalent bonds. These data are discussed with respect to biosynthesis and post-translational maturation of MPO as well as to its peculiar biochemical and biophysical properties.