Isopentenyldiphosphate:dimethylallyldiphosphate isomerase: construction of a high-level heterologous expression system for the gene from Saccharomyces cerevisiae and identification of an active-site nucleophile.
Isopentenyldiphosphate:dimethylallyldiphosphate isomerase (IPP isomerase) is an enzyme in isoprene metabolism which catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks for the pathway. The gene encoding IPP isomerase has recently been isolated from Saccharomyces cerevisiae [Anderson, M. S., Muehlbacher, M., Street, I.P., Proffitt, J., & Poulter, C. D. (1989) J. Biol. Chem. 264, 19169-19175]. A heterologous expression system was constructed for the gene and used to overexpress IPP isomerase in Escherichia coli. In transformants carrying the expression vector, IPP isomerase activity was increased by over 100,000-fold relative to that of the untransformed host strain. The overexpressed enzyme constitutes 30-35% of the total soluble cell protein and can be purified to homogeneity in two steps. Recombinant IPP isomerase was indistinguishable from that purified from yeast. 3-(Fluoromethyl)-3-butenyl diphosphate (FIPP) is a specific active-site-directed inhibitor of IPP isomerase from Claviceps purpurea [Muehlbacher, M., & Poulter, C. D. (1988) Biochemistry 27, 7315-7328]. Inactivation of yeast IPP isomerase by FIPP was active-site-directed, and inhibition resulted in formation of a stoichiometric enzyme-inhibitor complex. The site of covalent attachment in the enzyme-inhibitor complex was determined by inactivating IPP isomerase with [4-3H]FIPP, followed by digestion of the labeled enzyme with trypsin and purification of the resulting radioactive peptides by reversed-phase high-performance liquid chromatography. The primary site of attachment was Cys-139.
Street
I P
IP
Department of Chemistry, University of Utah, Salt Lake City 84112.
Poulter
C D
CD
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D019748:Carbon-Carbon Double Bond Isomerases; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D045782:Hemiterpenes; D007535:Isomerases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*
D004271:DNA, Fungal; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; C066270:3-(fluoromethyl)-3-butenyl diphosphate; D007535:Isomerases; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase