Purification of recombinant mandelate racemase: improved catalytic activity.
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.
Narmandakh
Ariun
A
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada B3H 1X5.
Bearne
Stephen L
SL
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
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