Human isopentenyl diphosphate: dimethylallyl diphosphate isomerase: overproduction, purification, and characterization.
Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. 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 [F.M. Hahn and C.D. Poulter (1995) J. Biol. Chem. 270, 11298-11303]. The human cDNA sequence was cloned into expression plasmid pFMH12. The encoded protein was overproduced in Escherichia coli and purified to > 90% homogeneity in two steps by ion-exchange and hydrophobic interaction chromatography. 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 microM, similar to the value of 43 microM reported for yeast IPP isomerase; Vmax = 4.1 mumol min-1 mg-1 for recombinant human IPP isomerase, approximately fivefold less than reported for the yeast enzyme [I.P. Street and C.D. Poulter (1990) Biochemistry 29, 7531-7538].
Hahn
F M
FM
Department of Chemistry, University of Utah, Salt Lake City 84112, USA.
Xuan
J W
JW
Chambers
A F
AF
Poulter
C D
CD
D000595:Amino Acid Sequence; D001483:Base Sequence; D019748:Carbon-Carbon Double Bond Isomerases; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D045782:Hemiterpenes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D045782:Hemiterpenes; D011994:Recombinant Proteins; D007535:Isomerases; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase