title: Purification of recombinant mandelate racemase: improved catalytic activity.
abstract: Mandelate racemase (MR, E.C. 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-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 pK(a) 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 (k(cat)) of 190+/-22 and 940+/-24s(-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+/-43s(-1). The MR fusion protein bearing an N-terminal StrepII-tag and a C-terminal decahistidine tag also exhibited reduced turnover (k(cat)=472+/-37s(-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.
authors: Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada B3H 1X5.
authors: Ariun
authors: A
authors: Narmandakh
authors: Stephen L
authors: SL
authors: Bearne
mesh_terms: D055162:Biocatalysis / Q000187:drug effects; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000378:metabolism; D007700:Kinetics; D009842:Oligopeptides / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017433:Protein Structure, Secondary; D016958:Pseudomonas putida / Q000201:enzymology*; D019998:Racemases and Epimerases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D019809:Streptavidin / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; D019809:Streptavidin; D013917:Thrombin; D019998:Racemases and Epimerases; C021540:mandelate racemase