Purification of recombinant mandelate racemase: Improved catalytic activity |
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| Authors: | Ariun Narmandakh Stephen L Bearne |
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| Institution: | 1. Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada B3H 1X5;2. Department of Chemistry, Dalhousie University, Halifax, NS, Canada B3H 4J3;1. Department of Inorganic and Analytical Chemistry, University of Szeged, Dóm tér 7, H-6720 Szeged, Hungary;2. Institute of Pharmaceutical Chemistry, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary;3. From the Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR),;4. Departamento de Microbiología, and;5. Departamento de Química Biológica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, Ocampo y Esmeralda, 2000-Rosario, Argentina;1. Department of Tourism and Leisure, Hsing Wu University, Taipei, 244, Taiwan;2. Research Center of Natural Cosmeceuticals Engineering, Xiamen Medical College, China;3. Department of Biological Science and Technology, China University of Science and Technology, Taipei, 115, Taiwan |
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| Abstract: | Mandelate racemase (MR, E.C. 5.1.2.2) from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate and has been studied extensively as a model for understanding how enzymes catalyze the deprotonation of carbon acid substrates with relatively high pKa values. Purification of recombinant MR as a fusion protein with an N-terminal hexahistidine tag using immobilized-nickel ion affinity chromatography and elution with a linear gradient of EDTA revealed three enzyme species (mrI, mrII, and mrIII). While mrIII was catalytically inactive, both mrI and mrII catalyzed the racemization of (S)-mandelate with turnover numbers (kcat) of 190 ± 22 and 940 ± 24 s?1, respectively. Circular dichroism analysis suggested that mrIII was a partially unfolded or misfolded form of the enzyme. Replacement of the N-terminal hexahistidine tag by a StrepII-tag appeared to ameliorate the folding problem yielding a single enzyme species with a turnover number of 1124 ± 43 s?1. The MR fusion protein bearing an N-terminal StrepII-tag and a C-terminal decahistidine tag also exhibited reduced turnover (kcat = 472 ± 37 s?1). These results highlight a potential problem that may be encountered when producing fusion enzymes bearing a polyhistidine tag: soluble, active enzyme may be obtained but care must be taken to ensure that it is free of minor misfolded forms that can alter the apparent activity of the enzyme. |
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