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 activat ion step in the isoprene biosynthetic pathway. The Saccharomyces cerev isiae gene for IPP isomerase, IDI1, was recently isolated and characte rized (Anderson, M. S., Muehlbacher, M., Street, I. P., Proffitt, J., and Poulter, C. D, (1989) J. Biol. Chem. 264, 19169-19175), and the wi ld-type gene, IDI1, was disrupted with a LEU2 marker to create a diplo id yeast strain heterozygous for the idi1::leu2 disruption, which reve aled 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). W e now report the isolation of a cDNA clone from Schizosaccharomyces po mbe by a plasmid shuffle-mediated complementation of the LEU2 disrupte d yeast gene. The S. pombe clone encoded a 26,864-dalton polypeptide o f 227 amino acids with a high degree of similarity to the S. cerevisia e IDI1 enzyme. S. pombe IPP isomerase contained the essential Cys and Glu catalytic residues identified in yeast isomerase (Street, I. P., C offman, H. R., Baker, J., and Poulter, C. (1994) Biochemistry 33, 4212 -4217) but was significantly smaller than the S. cerevisiae enzyme. Th e plasmid shuffle technique is an excellent procedure for screening ex pression libraries for IPP isomerase activity by complementation of th e idi1 mutation.