A generic HTS assay for kinase screening: Validation for the isolation of an engineered malate kinase
| Autor: | Isabelle André, Nelly Martineau, Audrey Baylac, Clément Auriol, Magali Remaud-Simeon, Jean-Marie François, Christopher M. Topham, Romain Irague, Thomas Walther |
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| Přispěvatelé: | 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 |
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
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 |
| Zdroj: | 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) |
| ISSN: | 1932-6203 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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