Hahn F M, Poulter C D
Department of Chemistry, University of Utah, Salt Lake City 84112, USA.
J Biol Chem. 1995 May 12;270(19):11298-303. doi: 10.1074/jbc.270.19.11298.
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.
异戊烯基二磷酸（IPP）异构酶催化异戊二烯生物合成途径中的一个关键激活步骤。酿酒酵母中IPP异构酶的基因IDI1最近已被分离和鉴定（安德森，M.S.，米尔巴赫，M.，斯特里特，I.P.，普罗菲特，J.，和波尔特，C.D.（1989年）《生物化学杂志》264卷，第19169 - 19175页），野生型基因IDI1被一个LEU2标记破坏，以创建一个对于idi1::leu2破坏杂合的二倍体酵母菌株，这表明IDI1是一个必需的单拷贝基因（迈耶，M.P.，哈恩，F.M.，斯蒂尔曼，D.J.，和波尔特，C.D.（1992年）《酵母》8卷，第743 - 748页）。我们现在报告通过质粒穿梭介导的对LEU2破坏的酵母基因的互补作用，从粟酒裂殖酵母中分离出一个cDNA克隆。粟酒裂殖酵母克隆编码一个由227个氨基酸组成、分子量为26,864道尔顿的多肽，与酿酒酵母IDI1酶具有高度相似性。粟酒裂殖酵母IPP异构酶含有在酵母异构酶中鉴定出的必需的半胱氨酸和谷氨酸催化残基（斯特里特，I.P.，科夫曼，H.R.，贝克，J.，和波尔特，C.（1994年）《生物化学》33卷，第4212 - 4217页），但明显小于酿酒酵母的酶。质粒穿梭技术是通过idi1突变的互补作用筛选IPP异构酶活性表达文库的一种出色方法。
