HUMAN ISOPENTENYL DIPHOSPHATE-DIMETHYLALLYL DIPHOSPHATE ISOMERASE - OVERPRODUCTION, PURIFICATION, AND CHARACTERIZATION

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase cata lyzes an essential activation step in the isoprenoid biosynthetic path way. A human cDNA sequence [J. Xuan, J. Kowalski, A. F. Chambers, and D. T. Denhardt (1994) Genomics 20, 129-131] containing a 684-base-pair open reading frame was recently reported that encoded a protein with a significant degree of similarity to two fungal IPP isomerases TF. M. Hahn and C. D. Poulter (1995) J. Biol. Chem. 270, 11298-11303]. The h uman cDNA sequence was cloned into expression plasmid pFMH12. The enco ded protein was overproduced in Escherichia coli and purified to >90% homogeneity in two steps by ion-exchange and hydrophobic interaction c hromatography. The recombinant protein catalyzed the isomerization of IPP to dimethylallyl diphosphate and was maximally active at pH 7.0 in the presence of Mg2+. The Michaelis constant for IPP was 33 mu M, sim ilar to the value of 43 mu M reported for yeast IPP isomerase; V-max = 4.1 mu mol min(-1) mg-l for recombinant human IPP isomerase, approxim ately fivefold less than reported for the yeast enzyme [L. P. Street a nd C. D. Poulter (1990) Biochemistry 29, 7531-7538]. (C) 1996 Academic Press, Inc.