Mandelate racemase (MR, E.C. 5.1.2.2) from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts the enantionners 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 +/- 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 Strepll-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 Strepll-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. (C) 2009 Elsevier Inc. All rights reserved.