Isopentenyl diphosphate:dimethylallyl diphosphate isomerase. An improved purification of the enzyme and isolation of the gene from Saccharomyces cerevisiae.
Isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IPP isomerase) is an enzyme in the isoprenoid biosynthetic pathway which catalyzes the interconversion of the primary five-carbon homoallylic and allylic diphosphate building blocks. We report a substantially improved procedure for purification of this enzyme from Saccharomyces cerevisiae. An amino-terminal sequence (35 amino acids) was obtained from a highly purified preparation of IPP isomerase. Oligonucleotide probes based on the protein sequence were used to isolate the structural gene encoding IPP isomerase from a yeast lambda library. The cloned gene encodes a 33,350-dalton polypeptide of 288 amino acids. A 3.5-kilobase EcoRI fragment containing the gene was subcloned into the yeast shuttle vector YRp17. Upon transformation with plasmids containing the insert, a 5-6-fold increase in IPP isomerase activity was seen in transformed cells relative to YRp17 controls, confirming the identity of the cloned gene. This is the first reported isolation of the gene for IPP isomerase.
Anderson
M S
MS
Department of Chemistry, University of Utah, Salt Lake City 84112.
Muehlbacher
M
M
Street
I P
IP
Proffitt
J
J
Poulter
C D
CD
D000595:Amino Acid Sequence; D001483:Base Sequence; D019748:Carbon-Carbon Double Bond Isomerases; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D005800:Genes, Fungal; D045782:Hemiterpenes; D007535:Isomerases / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D003062:Codon; D004271:DNA, Fungal; D045782:Hemiterpenes; D007535:Isomerases; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase