دورية أكاديمية
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: | Romain Irague, Christopher M Topham, Nelly Martineau, Audrey Baylac, Clément Auriol, Thomas Walther, Jean-Marie François, Isabelle André, Magali Remaud-Siméon |
| المصدر: | PLoS ONE, Vol 13, Iss 2, p e0193036 (2018) |
| بيانات النشر: | Public Library of Science (PLoS), 2018. |
| سنة النشر: | 2018 |
| المجموعة: | LCC:Medicine LCC:Science |
| مصطلحات موضوعية: | Medicine, Science |
| 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 (kcat/Km)malate/(kcat/Km)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. |
| نوع الوثيقة: | article |
| وصف الملف: | electronic resource |
| اللغة: | English |
| تدمد: | 1932-6203 |
| Relation: | http://europepmc.org/articles/PMC5819781?pdf=render; https://doaj.org/toc/1932-6203 |
| DOI: | 10.1371/journal.pone.0193036 |
| URL الوصول: | https://doaj.org/article/9a630c2d6ddf461a9b721db2b7d8872e |
| رقم الأكسشن: | edsdoj.9a630c2d6ddf461a9b721db2b7d8872e |
| قاعدة البيانات: | Directory of Open Access Journals |
Full Text Finder | تدمد: | 19326203 |
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| DOI: | 10.1371/journal.pone.0193036 |