17 research outputs found
Chemoenzymatic synthesis of polypeptides in neat 1,1,1,2-tetrafluoroethane solvent
Chemoenzymatic polypeptide synthesis offers several advantages over chemical or other biological routes, however, the use of aqueous-based media suffers from reverse hydrolysis reactions that challenge peptide chain propagation. Herein, the protease from subtilisin Carlsberg biocatalyzed the synthesis of poly-L-PheOEt, poly-L-LeuOEt, and the copolymers poly-L-PheOEt-co-L-LeuOEt from their amino acid ethyl ester substrates in a neat liquid 1,1,1,2-tetrafluoroethane solvent. The products, achieved in acceptable yields (ca. 50%), were fully characterized showing relatively high molar mass (ca. 20 000 Da for poly-L-PheOEt). This non-toxic low-boiling hydrofluorocarbon enhances enzymatic peptide propagation by limiting hydrolysis owing to its hydrophobic and relatively polar characteristics that sustain the protease activity and solubilize substrates and products. Computational molecular dynamic calculations were used to assess the L-PheOEt/L-LeuOEt-solvent and polypeptide-solvent interactions in this system. Additionally, the homopolypeptides displayed higher crystallinity than the copolypeptides with random incorporation of amino acid ethyl esters, notwithstanding the significantly highest specificity for Phe in this system. Interestingly, secondary structure characterization of the products by FTIR and circular dichroism suggests a non-common peptide folding
Inhibición de lipasa pancreática por flavonoides: importancia del doble enlace C2=C3 y la estructura plana del anillo C//Inhibition of pancreatic lipase by flavonoids: relevance of the C2=C3 double bond and C-ring planarity
Lipasa pancreática es una enzima clave en el metabolismo de lípidos. Los flavonoides son compuestos bioactivos de gran relevancia debido a sus interacciones con enzimas digestivas. Se evaluó la actividad de lipasa pancreática en presencia de flavonoides. Mediante espectroscopía UVVisible se determinó que el mejor inhibidor fue quercetina, seguido de rutina > luteolina > catequina > hesperetina, con valores de IC50 de 10.30, 13.50, 14.70, 28.50 y 30.50 μM, respectivamente. Todos los flavonoides mostraron una inhibición mixta, excepto catequina que mostró una inhibición acompetitiva. La capacidad inhibitoria de los flavonoides se relacionó con propiedades estructurales compartidas entre los distintos flavonoides, como la hidroxilación en las posiciones C5, C7 (anillo A), C2’ y C3’ (anillo B), y el doble enlace entre C2 y C3 (anillo C). Los resultados de inhibición coincidieron con el análisis de la fluorescencia extrínseca. Los estudios de docking molecular indicaron que la interacción entre lipasa pancreática y los flavonoides fue principalmente mediante interacciones hidrofóbicas (pi-stacking). Las interacciones de todos los flavonoides, excepto rutina, se dieron en el mismo sitio (subsitio 1) de la enzima. La insaturación entre C2 y C3 fue determinante para el acomodo de los flavonoides con la enzima, principalmente por interacciones de pi-stacking.ABSTRACTPancreatic lipase is a key enzyme in lipid metabolism. Flavonoids are bioactive compounds obtained from vegetables with big relevance, due to their intrinsic interaction with digestive enzymes. Pancreatic lipase activity was evaluated in the presence of flavonoids, through UV-Vis spectroscopy. All tested flavonoids showed a mixed-type inhibition, except catechin, which showed a uncompetitive inhibition. The best inhibitor was quercetin followed by rutin > luteolin > catechin > hesperetin, with IC50 values of 10.30, 13.50, 14.70, 28.50 and 30.50 μM, respectively. The flavonoids inhibitory capacity was related to structural properties shared between the different flavonoids, such as the hydroxylation at C5, C7 (ring A), C2’ and C3’ (ring B), and the double bond between C2 and C3 (ring C). The inhibition results are in agreement with the extrinsic fluorescence analysis. Molecular docking studies indicated that the interaction between pancreatic lipase and flavonoids was mainly through hydrophobic interactions (pi-stacking). The interactions of all flavonoids, except rutin, occurred at the same enzyme site (subsite 1). Instauration between C2 and C3 was decisive for the arrangement of flavonoids with the enzyme, mainly due to pi-stacking interactions
Estudio de la flexibilidad en el motivo ASA/[beta]B´/helice 5/ASA/[beta]C´(158-187) en la regulacion alosterica de la glucosamina-6-fosfato desaminasa de Escherichia coli
Enzyme-catalyzed processes environmentally friendly
Tema del mesLas enzimas son catalizadores biológicos que ofrecen un gran potencial de aplicación en los procesos químicos tradicionales. Éstas son altamente selectivas, funcionan adecuadamente en condiciones de reacción suaves y, por su naturaleza proteica, son amigables con el ambiente. Su aplicación en síntesis química ha permitido simplificar procesos, reducir la energía consumida en los mismos y disminuir la cantidad generada de residuos químicos contaminantes. Las enzimas también pueden funcionar en medios de reacción no convencionales, como disolventes orgánicos y fluidos supercríticos. Sus propiedades se pueden ajustar mediante la selección adecuada del medio de reacción, por inmovilización en soportes sólidos y por técnicas de diseño de proteínas y evolución dirigida. La inclusión de procesos catalizados por enzimas en las industrias química y farmacéutica está avanzando, beneficiándonos no sólo en el costo de los productos, sino en la generación de procesos más limpios y amigables con el ambiente.Enzymes are proteins, polymers composed by chemically bonded aminoacid molecules, which catalyze a wide range of chemicals reactions inside organisms. The catalytic activity of enzymes depends on their three-dimensional (3D) structure. Inside this 3D structures there are cavities, called “active site”, that shows affinity for specific molecules (called substrates) that will eventually become products. The combination of chemical functional groups in the active site creates covalent and non-covalent interactions between the protein and the substrate molecules; these interactions favor the conversion of substrates into products. As any catalyst, after the substrate transformation and once the product has been released from the active site, the enzyme returns to a basal or ground state and is ready to engage in a new catalytic cycle. Enzymes function outside cells, and historically men have used enzymes for their benefit. The most ancient enzyme applications are related to food, such as the production of bread and cheese. In this article we explain how efficient enzymes are and how do they work. We also present a brief account on the history of their discovery and the scientific breakthroughs that allowed the development of biocatalysis as one of the most significant biotechnologies in the modern era
Synthesis and modification of the Amyloid peptide sequence 37-42 of Aβ42 (AβPP): Efficient synthesis of N-methylated peptides, expanding the toolsfor peptide research
[EN] We report the synthesis and characterization of N-alkyl modified peptides by efficient coupling of N-methyl amino acids in solution phase. As a model peptide, the segment 37-42 (GGVVIA) of the Aβ-42 amyloid peptide derived from the amyloid precursor protein (Aβ-PP) was chosen. This peptide and its derivatives with N-methyl groups on Val40 and Ile41 residues were synthesized and character-ized. Because the synthesis was performed in solution-phase, the pro-cedure can be easily scaled up for the production of larger amounts of the peptides described in this work or any linear N-methyl peptide with potential therapeutic application.[ES] Se describe aquí la síntesis y caracterización de péptidos N-alquil modificados por acoplamiento eficiente en solución de N-Me-til aminoácidos. Se eligió como péptido modelo el segmento de 37-42 (GGVVIA) del péptido amiloide Aβ-42 derivado de la proteína pre-cursora de amiloide (Aβ-PP). Este péptido y sus derivados con grupos N-metilo en los residuos Val40 y Ile41 fueron sintetizados y caracteri-zados. Dado que ésta síntesis se realizó en solución, el procedimiento puede escalarse fácilmente y producir cantidades suficientes de los péptidos descritos, pudiendo aplicar ésta técnica en otros péptidos N-metilados lineales con posible potencial aplicación terapéutica.We are grateful to CONACYT for financial support (Project CB2010/151875); for scholarship to M.E.R-V. A. B. thanks the Plan Nacional de Investigación, MINECO, Spain and FSE funds for generous support through grant SAF2013-48399-R and Laboratorio Nacional de Estructura de Macromoléculas (LANEM, CONACyT 251613).Peer Reviewe
A novel approach to trypsin inhibition by flavonoids
Trypsin is a key protease related to digestion and absorption of proteins, which its inhibition must be studied
when natural compounds, such as flavonoids, are used as part of alternative treatments for obesity and diabetes
mellitus type 2, since trypsin and other pancreatic enzymes work at small intestine. Considering that flavonoids
are good lipase and amylase inhibitors, trypsin-flavonoids interactions were analyzed through UV-Vis, intrinsic
and extrinsic fluorescence spectroscopies, circular dichroism, and molecular docking. The interaction between
porcine pancreas trypsin and five flavonoids: hesperetin (HES), luteolin (LUT), quercetin (QUE), catechin (CAT),
and rutin (RUT) was evaluated. Most of them exhibited a mixed-type inhibition mode. LUT was the best trypsin
inhibitor (e.g., lower IC50, 45.20 ± 1.00 μM). All flavonoids-trypsin complexes showed static quenching, and QUE
and LUT exhibited higher affinity (associative binding constant, Ka values, 0.90 ± 0.10 and 1.60 ± 0.20·10−1 mM−1,
respectively). Hydrophobic interactions between trypsin and flavonoids were predominant
Immobilization and Biochemical Properties of the Enantioselective Recombinant NStcI Esterase of Aspergillus nidulans
The recombinant NStcI A. nidulans esterase was adsorbed on Accurel MP1000, where protein yield and immobilization efficiency were 42.48% and 81.94%, respectively. Storage stability test at 4°C and RT showed 100% of residual activity after 40 days at both temperatures. The biocatalyst retains more than 70% of its initial activity after 3 cycles of repeated use. Biochemical properties of this new biocatalyst were obtained. Maximum activity was achieved at pH 11 and 30°C, while the best stability was observed with the pH between 9 and 11 at 40°C. NStcI thermostability was increased after immobilization, as it retained 47.5% of its initial activity after 1 h at 60°C, while the free enzyme under the same conditions displayed no activity. NStcI preserved 70% of its initial activity in 100% hexane after 72 h. Enzymatic kinetic resolution of (R,S)-1-phenylethanol was chosen as model reaction, using vinyl acetate as acyl donor. After optimization of reaction parameters, the highest possible conversion (42%) was reached at 37°C, aw of 0.07, and 120 h of bioconversion in hexane with an enantiomeric excess of 71.7%. NStcI has selectivity for (R)-enantiomer. The obtained E value (31.3) is in the range considered useful to resolve enantiomeric mixtures
Interaction of N-succinyl diaminopimelate desuccinylase with orphenadrine and disulfiram
The emergence of multiresistant, often persistent, pathogenic bacteria emphasizes the need for a continuous identification of new pharmacological targets of enzymatic nature, and the development of selective inhibitors against them. Ubiquitously present in most bacteria, the enzyme metallohydrolase N-succinyl diaminopimelate desuccinylase (DapE) is required for the biosynthesis of meso-diaminopimelate (mDAP) and lysine, both essential components of bacterial peptidoglycan. DapE activity has been recognized as critical for bacterial growth; and thus, it is a potential pharmacological target. In order to develop effective inhibitors against DapE, understanding of structural and functional features of the enzyme must be used in the design, such as the interaction of its metal centers with ligands, as well as its effect on global protein conformational changes that seem to produce an induced fit after ligand binding. Here, we propose the potential application of currently approved drugs, used in different medical fields, orphenadrine and disulfiram, as possible inhibitory compounds against DapE, based on studies of equilibrium ligand binding, inhibition, thermal stability and molecular docking into Enterococcus faecium and Escherichia coli DapE enzyme homologs. Drugs were selected based on key structural features, including the presence of soft heteroatoms or π-bonds that are known to interact with DapE active site. Enzymes from selected bacteria were chosen based on the pattern of infection, persistence, and drug resistance as well to study an enzyme from the two Gram classification. Furthermore, the information presented here can further provide structural details about key interacting functional groups, which should be considered in the design and development of a new generation of antibiotics that can target the essential DapE enzyme
Relevance of aromatic and polar amino acids in the specificity of Inulinase ISO3 from Kluyveromyces marxianus: A molecular dynamics approach with an experimental verification
11 pags., 8 figs., 5 tabs.The Inulinase from Kluyveromyces marxianus ISO3 (Inu-ISO3) is an enzyme able to hydrolyze linear fructans such as chicory inulin as well as branched fructans like agavin. This enzyme was cloned and expressed in Komagataella pastoris to study the role of selected aromatic and polar residues in the catalytic pocket by Alanine scanning. Molecular dynamics (MD) simulations and enzyme kinetics analysis were performed to study the functional consequences of these amino acid substitutions. Site-directed mutagenesis was used to construct the mutants of the enzyme after carrying out the MD simulations between Inu-ISO3 and its substrates. Mutation Trp79:Ala resulted in the total loss of activity when fructans were used as substrates, while with sucrose, the activity decreased by 98 %. In contrast, the mutations Phe113:Ala and Gln236:Ala increased the invertase activity when sucrose was used as a substrate. Although these amino acids are not part of the conserved motifs where the catalytic triad is located, they are essential for the enzyme's activity. In silico and experimental approaches corroborate the relevance of these residues for substrate binding and their influence on enzymatic activity.This work was supported by DGAPA-UNAM (Grant PAPIIT IN219520). J. Trapala acknowledges financial support from CONACyT through a Ph.D scholarship.Peer reviewe