A generic HTS assay for kinase screening: Validation for the isolation of an engineered malate kinase
| العنوان: | A generic HTS assay for kinase screening: Validation for the isolation of an engineered malate kinase |
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| المؤلفون: | Isabelle André, Nelly Martineau, Audrey Baylac, Clément Auriol, Magali Remaud-Simeon, Jean-Marie François, Christopher M. Topham, Romain Irague, Thomas Walther |
| المساهمون: | Toulouse White Biotechnology (TWB), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), French National Research Agency (ANR programme d'Investissement d'Avenir, Project SYNTHACS) [ANR-10-BTBR-05-01], ANR-10-BTBR-0005,SYNTHACS,Biologie Synthétique pour la synthèse de molécules chimiques à haute valeur ajoutée à partir de ressources carbonées renouvelables(2010), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Andre, Isabelle, Remaud Simeon, Magali |
| المصدر: | PLoS ONE PLoS ONE, Public Library of Science, 2018, 13 (2), 15 p. ⟨10.1371/journal.pone.0193036⟩ PLoS ONE, 2018, 13 (2), 15 p. ⟨10.1371/journal.pone.0193036⟩ PLoS ONE, Vol 13, Iss 2, p e0193036 (2018) Plos One 2 (13), 15 p.. (2018) |
| بيانات النشر: | HAL CCSD, 2018. |
| سنة النشر: | 2018 |
| مصطلحات موضوعية: | 0301 basic medicine, Models, Molecular, enzymic activity, métabolisme du malate, Applied Microbiology, [SDV]Life Sciences [q-bio], Mutant, Malates, lcsh:Medicine, Protein Engineering, nicotinamide-adenine dinucleotide, 01 natural sciences, Biochemistry, nad, Substrate Specificity, Electricity, Catalytic Domain, Enzyme Inhibitors, lcsh:Science, Multidisciplinary, 010304 chemical physics, Kinase, Chemistry, Physics, activité enzymatique, Recombinant Proteins, Enzymes, Bioassays and Physiological Analysis, Physical Sciences, Engineering and Technology, Research Article, Biotechnology, Static Electricity, Library Screening, Research and Analysis Methods, Microbiology, Catalysis, 03 medical and health sciences, Industrial Microbiology, Electrostatics, 0103 physical sciences, Aspartate kinase, Enzyme kinetics, Aspartate Kinase, Kinase activity, Molecular Biology Techniques, Molecular Biology, Enzyme Assays, Gene Library, adénosine di phosphate, Molecular Biology Assays and Analysis Techniques, lcsh:R, Phosphotransferases, Substrate (chemistry), Biology and Life Sciences, Proteins, Genetic Variation, High-Throughput Screening Assays, Kinetics, 030104 developmental biology, adenosine pyrophosphate, Amino Acid Substitution, criblage, Enzymology, Biocatalysis, Mutagenesis, Site-Directed, lcsh:Q, NAD+ kinase, Directed Molecular Evolution, Biochemical Analysis, size grading, Pyruvate kinase, Cloning |
| الوصف: | An end-point ADP/NAD(+) acid/alkali assay procedure, directly applicable to library screening of any type of ATP-utilising/ADP producing enzyme activity, was implemented. Typically, ADP production is coupled to NAD(+) co-enzyme formation by the conventional addition of pyruvate kinase and lactate dehydrogenase. Transformation of enzymatically generated NAD(+) into a photometrically active alkali derivative product is then achieved through the successive application of acidic/alkali treatment steps. The assay was successfully miniaturized to search for malate kinase activity in a structurally-guided library of LysC aspartate kinase variants comprising 6,700 clones. The screening procedure enabled the isolation of nine positive variants showing novel kinase activity on (L)-malate, the best mutant, LysC V115A: E119S:E434V exhibited strong substrate selectivity for (L)-malate compared to (L)-aspartate with a (k(cat)/K-m)(malate)/(k(cat)/K-m)(aspartate) ratio of 86. Double mutants V115A:E119S, V115A:E119C and E119S:E434V were constructed to further probe the origins of stabilising substrate binding energy gains for (L)-malate due to mutation. The introduction of less sterically hindering side-chains in engineered enzymes carrying E119S and V115A mutations increases the effective volume available for substrate binding in the catalytic pocket. Improved binding of the (L)-malate substrate may be assisted by less hindered movement of the Phe184 aromatic side-chain. Additional favourable long-range electostatic effects on binding arising from the E434V surface mutation are conditionally dependent upon the presence of the V115A mutation close to Phe184 in the active-site. |
| وصف الملف: | application/pdf |
| اللغة: | English |
| تدمد: | 1932-6203 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::29381b9349a19b8604e314ec1ddd02c3 https://hal.archives-ouvertes.fr/hal-01886455/file/journal.pone.0193036.pdf |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....29381b9349a19b8604e314ec1ddd02c3 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 19326203 |
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