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Vidossich P.,Laboratori Of Simulacio Computacional I Modelitzacio | Vidossich P.,Institute Of Quimica Teorica I Computacional Iqtcub | Carpena X.,Barcelona Institute for Research in Biomedicine | Loewen P.C.,University of Manitoba | And 2 more authors.
Journal of Physical Chemistry Letters | Year: 2011

By means of quantum mechanics/molecular mechanics calculations, we show that binding of dioxygen to the FeIII enzyme catalase-peroxidase (KatG), responsible for activating the antitubercular drug isoniazid, is possible in the absence of an external reducing agent, thanks to the unique electronic properties of the active site Met-Tyr-Trp adduct. The calculations give support to recent experimental observations suggesting that KatG activates molecular oxygen and suggest that dioxygen activation may be achieved in other enzymes by inserting a residue with low ionization potential near the active site. © 2011 American Chemical Society. Source

Ardevol A.,Institute Of Quimica Teorica I Computacional Iqtcub | Biarnes X.,Ramon Llull University | Planas A.,Ramon Llull University | Rovira C.,Institute Of Quimica Teorica I Computacional Iqtcub | Rovira C.,Catalan Institution for Research and Advanced Studies
Journal of the American Chemical Society | Year: 2010

The mechanism of glycosidic bond cleavage by glycosidases involves substrate ring distortions in the Michaelis complex that favor catalysis. Retaining β-mannosidases bind the substrate in a 1S5 conformation, and recent experiments have proposed an unusual substrate conformational pathway (1S5 → B2,5 → OS2) for the hydrolysis reaction. By means of Car-Parrinello metadynamics simulations, we have obtained the conformational free-energy surface (FES) of a β-d-mannopyranose molecule associated with the ideal Stoddart conformational diagram. We have found that 1S 5 is among the most stable conformers and simultaneously is the most preactivated conformation in terms of elongation/shortening of the C1-O1/C1-O5 bonds, C1-O1 orientation, and charge development at the anomeric carbon. Analysis of the computed FES gives support to the proposed 1S 5 → B2,5 → OS2 catalytic itinerary, showing that the degree of preactivation of the substrate in glycoside hydrolases (GHs) is related to the properties of an isolated sugar ring. We introduce a simple preactivation index integrating several structural, electronic, and energetic properties that can be used to predict the conformation of the substrate in the Michaelis complex of any GH. © 2010 American Chemical Society. Source

Alfonso-Prieto M.,Computer Simulation and Modeling Laboratory CosMoLab | Kumar M.,University of Louisville | Rovira C.,Computer Simulation and Modeling Laboratory CosMoLab | Rovira C.,Institute Of Quimica Teorica I Computacional Iqtcub | And 2 more authors.
Journal of Physical Chemistry B | Year: 2010

The key step in the catalytic cycle of methionine synthase (MetH) is the transfer of a methyl group from the methylcobalamin (MeCbl) cofactor to homocysteine (Hcy). This mechanism has been traditionally viewed as an S N2-type reaction, but a different mechanism based on one-electron reduction of the cofactor (reductive cleavage) has been recently proposed. In this work, we analyze whether this mechanism is plausible from a theoretical point of view. By means of a combination of gas-phase as well as hybrid QM/MM calculations, we show that cleavage of the Co - C bond in a MeCbl· ··Hcy complex (Hcy = methylthiolate substrate (Me-S-), a structural mimic of deprotonated homocysteine) proceeds via a [Co III(corriṅ-)] - Me··· ̇S-Me diradical configuration, involving electron transfer (ET) from a π*corrin-type state to a σ* Co - C one, and the methyl transfer displays an energy barrier ≤8.5 kcal/mol. This value is comparable to the one previously computed for the alternative SN2 reaction pathway (10.5 kcal/mol). However, the ET-based reductive cleavage pathway does not impose specific geometrical and distance constraints with respect to substrate and cofactor, as does the S N2 pathway. This might be advantageous from the enzymatic point of view because in that case, a methyl group can be transferred efficiently at longer distances. © 2010 American Chemical Society. Source

Ruiz F.X.,Autonomous University of Barcelona | Porte S.,Autonomous University of Barcelona | Gallego O.,Autonomous University of Barcelona | Moro A.,Autonomous University of Barcelona | And 8 more authors.
Biochemical Journal | Year: 2011

Human AKR (aldo-keto reductase) 1C proteins (AKR1C1-AKR1C4) exhibit relevant activity with steroids, regulating hormone signalling at the pre-receptor level. In the present study, investigate the activity of the four human AKR1C enzymes with retinol and retinaldehyde. All of the enzymes except AKR1C2 showed retinaldehyde reductase activity with low Km values (∼1 μM). The kcat values were also low (0.18-0.6 min -1), except for AKR1C3 reduction of 9-cis-retinaldehyde whose k cat was remarkably higher (13 min-1). Structural modelling of the AKR1C complexes with 9-cis-retinaldehyde indicated a distinct conformation of Trp227, caused by changes in residue 226 that may contribute to the activity differences observed. This was partially supported by the kinetics of the AKR1C3 mutant. Retinol/retinaldehyde conversion, combined with the use of the inhibitor flufenamic acid, indicated a relevant role for endogenous AKR1Cs in retinaldehyde reduction in MCF-7 breast cancer cells. Overexpression of AKR1C proteins depleted RA (retinoic acid) transactivation in HeLa cells treated with retinol. Thus AKR1Cs may decrease RA levels in vivo. Finally, by using lithocholic acid as an AKR1C3 inhibitor and UVI2024 as an RA receptor antagonist, we provide evidence that the pro-proliferative action of AKR1C3 in HL-60 cells involves the RA signalling pathway and that this is in part due to the retinaldehyde reductase activity of AKR1C3. © The Authors Journal compilation © 2011 Biochemical Society. Source

Vidossich P.,Autonomous University of Barcelona | Alfonso-Prieto M.,Temple University | Rovira C.,Laboratori Of Simulacio Computacional I Modelitzacio Cosmolab | Rovira C.,Institute Of Quimica Teorica I Computacional Iqtcub | Rovira C.,Catalan Institution for Research and Advanced Studies
Journal of Inorganic Biochemistry | Year: 2012

Catalases and peroxidases are ubiquitous heme enzymes that catalyze the removal of hydrogen peroxide (H2O2). Both enzymes use one molecule of hydrogen peroxide to form a high valent iron intermediate named Compound I (Cpd I). However, whereas catalase Cpd I oxidizes a second H 2O2 molecule to oxygen, peroxidases use this intermediate to oxidize other substrates rather than H2O2. The origin of the different reactivity of peroxidases and catalases is not known, but it is likely to be related to structural differences between the two heme active sites. Recent modeling studies suggest that the oxidation of H2O 2 by catalase Cpd I may take place by two hydrogen atom transfer steps. In this work, we investigate how catalases and peroxidases compare along the same hydrogen transfer steps to give hints into the question why peroxidases cannot efficiently oxidize H2O2. The use of simplified models allows us to probe the direct effect of the proximal ligand (tyrosinate in catalases and histidine in peroxidases) without masking from the protein environment. We show that the nature of the fifth ligand (His in peroxidase and Tyr in catalase) has little effect on the energy barriers of the hydrogen transfer steps. On the contrary, the Cpd I-hydrogen peroxide (O Fe-Operoxide) distance affects significantly the reaction barriers. We propose that the distal side architecture of peroxidases do not allow to attain short OCpd I-Operoxide distances, thus resulting in a lower efficiency towards H2O2 oxidation. © 2012 Elsevier Inc. All rights reserved. Source

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