Computational strategies for the design of new enzymatic functions

In this contribution, recent developments in the design of biocatalysts are reviewed with particular emphasis in the de novo strategy. Studies based on three different reactions, Kemp elimination, Diels–Alder and Retro-Aldolase, are used to illustrate different success achieved during the last years. Finally, a section is devoted to the particular case of designed metalloenzymes. As a general conclusion, the interplay between new and more sophisticated engineering protocols and computational methods, based on molecular dynamics simulations with Quantum Mechanics/Molecular Mechanics potentials and fully flexible models, seems to constitute the bed rock for present and future successful design strategies.This work was supported by the Spanish Ministerio de Economía y Competitividad for project CTQ2012-36253-C03, Universitat Jaume I – Spain (project P1·1B2014-26), Generalitat Valenciana – Spain (PROMETEOII/2014/022 and ACOMP/2014/277 projects), Polish National Center for Science (NCN) (grant 2011/02/A/ST4/00246, 2012−2017), the Polish Ministry of Science and Higher Education (“Iuventus Plus” program project no. 0478/IP3/2015/73, 2015-2016) and the USA National Institute of Health (ref. NIH R01 GM065368). Authors acknowledge computational resources from the Servei d’Informàtica of Universitat de València on the ‘Tirant’ supercomputer and the Servei d’Informat̀ica of Universitat Jaume I

Theoretical studies of the hydrolysis of antibiotics catalyzed by a metallo-B-lactamase

In this paper, hybrid QM/MM molecular dynamics (MD) simulations have been performed to explore the mechanisms of hydrolysis of two antibiotics, Imipenen (IMI), an antibiotic belonging to the subgroup of carbapenems, and the Cefotaxime (CEF), a third-generation cephalosporin antibiotic, in the active site of a mono-nuclear β- lactamase, CphA from Aeromonas hydrophila. According to our results, significant different transition state structures are obtained for the hydrolysis of both antibiotics: while the TS of the CEF is a ionic species with negative charge on nitrogen, the IMI TS presents a tetrahedral-like character with negative charge on oxygen atom of the carbonyl group of the lactam ring. Thus, dramatic conformational changes can take place in the cavity of CphA to accommodate different substrates, which would be the origin of its substrate promiscuity. This feature of the β-lactamase would be in turn, associated to the different mechanisms that the protein employs to hydrolyze the different antibiotics; i.e. the catalytic promiscuity. Since CphA shows only activity against carbapenem antibiotic, this study will be used to shed some light into the origin of the selectivity of the different MbL and, as a consequence, into the discovery of specific and potent MβL inhibitors against a broad spectrum of bacterial pathogens.We thank FEDER and the Spanish Ministerio de Economía y Competitividad (project CTQ2012-36253-C03), Universitat Jaume I (project P1 1B2011-23) and Generalitat Valenciana (PROMETEOII/2014/022 and ACOMP/2014/277 projects

Computational design of biological catalysts

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 catalyst

Molecular Mechanism of the site-specific self-cleavage of the RNA phosphodiester backbone by a Twister Ribozyme

Published as part of the special collection of articles derived from the 10th Congress on Electronic Structure: Principles and Applications (ESPA-2016).The catalytic activity of some classes of natural RNA, named as ribozymes, has been discovered just in the past decades. In this paper, the cleavage of the RNA phosphodiester backbone has been studied in aqueous solution and in a twister ribozyme from Oryza sativa. The free energy profiles associated with a baseline substrate-assisted mechanism for the reaction in the enzyme and in solution were computed by means of free energy perturbation methods within hybrid QM/MM potentials, describing the chemical system by the M06-2× functional and the environment by means of the AMBER and TIP3P force fields. The results confirm that this is a stepwise mechanism kinetically controlled by the second step that involves the P–O5′ breaking bond concomitant with the proton transfer from the OP1 atom to the leaving O5′ atom. 18O kinetic isotope effects on the nucleophile and leaving oxygen atoms, in very good agreement with experiments, also support this description. Nevertheless, the free energy profiles in the enzyme and in solution are almost coincident which, despite that the rate-limiting activation free energy is in very good agreement with experimental data of counterpart reactions in solution, rule out this substrate-assisted catalysis mechanism for the twister ribozyme from O. sativa. Catalysis must come from the role of alternative acid–base species not available in aqueous solution, but the rate-limiting transition state must be associated with the P–O5′ bond cleavage

Do zwitterionic species exist in the non-enzymatic peptide bond formation?

The use of proper computational methods and models has allowed answering the controversial question of whether zwitterionic species exist in the mechanism of peptide bond synthesis in aqueous solution. In fact, the different conformations of zwitterionic species open the door to different mechanistic paths

Diseño computacional de catalizadores biológicos

Enzymes are a source of inspiration for the design of new and powerful industrial catalysts able to work, in principle, in mild conditions of pressure, temperature and solvents. The Transition State of the reaction is the keystone to understand how enzymes are able to speed up reactions. This information can be then employed to design protein structures or successful mutations in the active site of proteins leading to the synthesis of new biocatalysts. In this paper we show advances in this field, classified according to the starting material: catalytic antibodies, modified enzymes and enzymes designed de novo.Las enzimas son una fuente de inspiración para el diseño de nuevos catalizadores con usos industriales que puedan funcionar en condiciones suaves de presión, temperatura y disolventes. El estado de transición de la reacción que se pretende catalizar, es la pieza clave para comprender cómo las enzimas son capaces de acelerar las reacciones. A partir de esta información se pueden proponer estructuras proteicas, o posibles mutaciones en el centro activo de proteínas, conduciendo a la síntesis de nuevos biocatalizadores. En este artículo presentamos los avances en este campo, clasificados en función del material de partida: anticuerpos catalíticos, enzimas modificadas y enzimas diseñadas de novo

Cuba, bienes funerarios y publicidad registral. Propuesta para su perfeccionamiento

El presente artículo está enmarcado en el estudio de los bienes funerarios con el fin de redimen-sionar los mecanismos de publicidad de que están dotados los mismos, teniendo como máxima representación de aquella, la creación de una sección dentro del Registro de la Propiedad, que se encargue de brindar una protección eficaz, cautelosa y rigurosa de dichos títulos, bajo la obser-vancia de los principios generales del derecho

Fructose metabolism in Chromohalobacter salexigens: interplay between the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways

Background The halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner–Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner–Doudoroff and Embden–Meyerhof–Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens. Results Fructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner–Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden–Meyerhof–Parnas (EMP) route. Tracking isotopic label from [1-13C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as fructose bisphosphatase, although it had been annotated previously as pyrophosphate-dependent phosphofructokinase. The gluconeogenic rather than glycolytic role of this enzyme in vivo is in agreement with the lack of 6-phosphofructokinase activity previously described. Conclusions Overall, this study shows that C. salexigens possesses a greater metabolic flexibility for fructose catabolism, the ED and EMP pathways contributing to a fine balancing of energy and biosynthetic demands and, subsequently, to a more efficient metabolism.University of Murcia and University of Seville was supported by projects: BIO2015-63949-R, BIO2014-54411-C2-1-REuropa MINECO/FEDER RTI2018-094393-B-C21Fundación Séneca (Grant no. 19236/PI/14

Poema musico alegorico, intitulado Paz entre Marte, y Cupido ... que la Real Maestranza ... de Valencia ha de celebrar en obsequio del ... Cumpleaños de ... Carlos III ...

Pie de imp. tomado de colofón.Sign. : [A]-B4, C5Friso tip. en p. 5, 17 y grab xil. de estrellas p. 16.Sin reclamos

Mendelian Randomization Analysis of the Relationship Between Native American Ancestry and Gallbladder Cancer Risk

Background A strong association between the proportion of Native American ancestry and the risk of gallbladder cancer (GBC) has been reported in observational studies. Chileans show the highest incidence of GBC worldwide, and the Mapuche are the largest Native American people in Chile. We set out to investigate the causal association between Native American Mapuche ancestry and GBC risk, and the possible mediating effects of gallstone disease and body mass index (BMI) on this association. Methods Markers of Mapuche ancestry were selected based on the informativeness for assignment measure and then used as instrumental variables in two-sample mendelian randomization (MR) analyses and complementary sensitivity analyses. Result We found evidence of a causal effect of Mapuche ancestry on GBC risk (inverse variance-weighted (IVW) risk increase of 0.8% for every 1% increase in Mapuche ancestry proportion, 95% CI 0.4% to 1.2%, p = 6.6×10-5). Mapuche ancestry was also causally linked to gallstone disease (IVW risk increase of 3.6% per 1% increase in Mapuche proportion, 95% CI 3.1% to 4.0%, p = 1.0×10-59), suggesting a mediating effect of gallstones in the relationship between Mapuche ancestry and GBC. In contrast, the proportion of Mapuche ancestry showed a negative causal effect on BMI (IVW estimate -0.006 kg/m2 per 1% increase in Mapuche proportion, 95% CI -0.009 to -0.003, p = 4.4×10-5). Conclusions The results presented here may have significant implications for GBC prevention and are important for future admixture mapping studies. Given that the association between Mapuche ancestry and GBC risk previously noted in observational studies appears to be causal, primary and secondary prevention strategies that take into account the individual proportion of Mapuche ancestry could be particularly efficient