Human isopentenyl diphosphate:dimethylallyl diphosphate isomerase: Overproduction, purification, and characterization
Human isopentenyl diphosphate:dimethylallyl diphosphate isomerase: Overproduction, purification, and characterization
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DOI：
10.1006/abbi.1996.0312
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发表时间：
1996-08-01
影响因子：
3.9
通讯作者：
Poulter, CD
中科院分区：
文献类型：
--
作者：
Hahn, FM;Xuan, JW;Poulter, CD
异戊烯基二磷酸(IPP)：二甲基烯丙基二磷酸异构酶催化类异戊二烯生物合成途径中的一个重要激活步骤。最近报道了一个含有684个碱基对的开放阅读框的人的cDNA序列[J.Xuan，J.Cotalski，A.F.Chambers，and D.T.Denhardt(1994)Genology 20,129-131]，该序列编码的蛋白质与真菌的两种IPP异构酶Tf有很大的相似性。M.Hahn和C.D.Poulter(1995)J.Biol.化学。11298-11303]。将人的基因克隆到表达载体pFMH12中。通过离子交换和疏水作用层析，在大肠杆菌中高效表达编码蛋白，两步纯化后纯度达到90%。该重组蛋白催化IPP异构化为二甲基烯丙基二磷酸酯，并在pH为7.0时有最大活性。重组人IPP异构酶的米氏常数V-max=4.1mU·min~(-1)mg·L，比酵母酶的米氏常数小近五倍[L.P.Street和C.D.Poulter(1990)BioChemical 29,7531-7538]。(C)1996年学术出版社。
Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A human cDNA sequence [J. Xuan, J. Kowalski, A. F. Chambers, and D. T. Denhardt (1994) Genomics 20, 129-131] containing a 684-base-pair open reading frame was recently reported that encoded a protein with a significant degree of similarity to two fungal IPP isomerases TF. M. Hahn and C. D. Poulter (1995) J. Biol. Chem. 270, 11298-11303]. The human cDNA sequence was cloned into expression plasmid pFMH12. The encoded protein was overproduced in Escherichia coli and purified to >90% homogeneity in two steps by ion-exchange and hydrophobic interaction chromatography. The recombinant protein catalyzed the isomerization of IPP to dimethylallyl diphosphate and was maximally active at pH 7.0 in the presence of Mg2+. The Michaelis constant for IPP was 33 mu M, similar to the value of 43 mu M reported for yeast IPP isomerase; V-max = 4.1 mu mol min(-1) mg-l for recombinant human IPP isomerase, approximately fivefold less than reported for the yeast enzyme [L. P. Street and C. D. Poulter (1990) Biochemistry 29, 7531-7538]. (C) 1996 Academic Press, Inc.