Isolation of Schizosaccharomyces pombe isopentenyl diphosphate isomerase cDNA clones by complementation and synthesis of the enzyme in Escherichia coli.
Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprene biosynthetic pathway. The Saccharomyces cerevisiae gene for IPP isomerase, IDI1, was recently isolated and characterized (Anderson, M. S., Muehlbacher, M., Street, I. P., Proffitt, J., and Poulter, C. D. (1989) J. Biol. Chem. 264, 19169-19175), and the wild-type gene, IDI1, was disrupted with a LEU2 marker to create a diploid yeast strain heterozygous for the idi1::leu2 disruption, which revealed that IDI1 was an essential single-copy gene (Mayer, M.P., Hahn, F. M., Stillman, D. J., and Poulter, C. D. (1992) Yeast 8, 743-748). We now report the isolation of a cDNA clone from Schizosaccharomyces pombe by a plasmid shuffle-mediated complementation of the LEU2 disrupted yeast gene. The S. pombe clone encoded a 26,864-dalton polypeptide of 227 amino acids with a high degree of similarity to the S. cerevisiae IDI1 enzyme. S. pombe IPP isomerase contained the essential Cys and Glu catalytic residues identified in yeast isomerase (Street, I. P., Coffman, H. R., Baker, J., and Poulter, C. (1994) Biochemistry 33, 4212-4217) but was significantly smaller than the S. cerevisiae enzyme. The plasmid shuffle technique is an excellent procedure for screening expression libraries for IPP isomerase activity by complementation of the idi1 mutation.
Hahn
F M
FM
Department of Chemistry, University of Utah, Salt Lake City 84112, USA.
Poulter
C D
CD
D000595:Amino Acid Sequence; D001483:Base Sequence; D019748:Carbon-Carbon Double Bond Isomerases; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D004926:Escherichia coli; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D045782:Hemiterpenes; D006801:Humans; D007535:Isomerases / Q000096:biosynthesis* / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D045782:Hemiterpenes; D011994:Recombinant Proteins; D007535:Isomerases; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase