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 p K 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 ± 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 ( k cat = 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.