Virtual screening of mandelate racemase mutants with enhanced activity based on binding energy in the transition state |
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| Affiliation: | 1. School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, PR China;2. Experiment Center for Science and Technology, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, PR China;3. Institute of Chinese Material Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, PR China;1. School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China;2. Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China;3. School of Pharmacy, Jinzhou Medical University, Jinzhou 121001, China;4. Testing Laboratory, Qinghai Entry-Exit Inspection and Quarantine Bureau, Xining, 810000,China;1. Ruprecht-Karls-Universität Heidelberg, Physikalisch-Chemisches Institut, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany;2. KIT – Campus Nord, Institut für Nukleare Entsorgung, P.O. Box 3640, 76021 Karlsruhe, Germany;3. Central Radionuclide Laboratory, Technische Universität Dresden, 01062 Dresden, Germany;4. Dipartimento di Scienze Chimiche, dell ‘Universitá di Padova, Via Marzolo, 1, 35131 Padova, Italy;1. Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;2. Division of Molecular Biology, Department of General, Visceral, Vascular and Thoracic Surgery, Charité – Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany |
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| Abstract: | Mandelate racemase (MR) is a promising candidate for the dynamic kinetic resolution of racemates. However, the poor activity of MR towards most of its non-natural substrates limits its widespread application. In this work, a virtual screening method based on the binding energy in the transition state was established to assist in the screening of MR mutants with enhanced catalytic efficiency. Using R-3-chloromandelic acid as a model substrate, a total of 53 mutants were constructed based on rational design in the two rounds of screening. The number of mutants for experimental validation was brought down to 17 by the virtual screening method, among which 14 variants turned out to possess improved catalytic efficiency. The variant V26I/Y54V showed 5.2-fold higher catalytic efficiency (kcat/Km) towards R-3-chloromandelic acid than that observed for the wild-type enzyme. Using this strategy, mutants were successfully obtained for two other substrates, R-mandelamide and R-2-naphthylglycolate (V26I and V29L, respectively), both with a 2-fold improvement in catalytic efficiency. These results demonstrated that this method could effectively predict the trend of mutational effects on catalysis. Analysis from the energetic and structural assays indicated that the enhanced interactions between the active sites and the substrate in the transition state led to improved catalytic efficiency. It was concluded that this virtual screening method based on the binding energy in the transition state was beneficial in enzyme rational redesign and helped to better understand the catalytic properties of the enzyme. |
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| Keywords: | Virtual screening Mandelate racemase Binding energy Transition state Molecular dynamics simulation |
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