Schulbach M C, Brennan P J, Crick D C
Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523, USA.
J Biol Chem. 2000 Jul 28;275(30):22876-81. doi: 10.1074/jbc.M003194200.
We report the cloning, overexpression, and partial characterization of two unique Z-isoprenyl diphosphate synthase homologs from Mycobacterium tuberculosis. The first enzyme, Rv1086, adds one isoprene unit to omega,E-geranyl diphosphate. The product, omega,E, Z-farnesyl diphosphate, is the putative substrate of the second enzyme, Rv2361c. This enzyme adds seven more isoprene units to omega, E,Z-farnesyl diphosphate and releases decaprenyl diphosphate. Both open reading frames were cloned from the M. tuberculosis H37Rv genome and overexpressed in M. smegmatis. Membrane and cytosol fractions from wild type and the two recombinant strains were assayed for [(14)C]isopentenyl diphosphate incorporation into isoprenyl diphosphates in the presence of various allylic isoprenyl diphosphate acceptors. Membrane fractions of recombinant cells overexpressing Rv2361c incubated with farnesyl diphosphate showed a 10-fold increase of [(14)C]isopentenyl diphosphate incorporation into decaprenyl diphosphate. Membrane fractions of recombinant cells overexpressing Rv1086 incubated with geranyl diphosphate showed a 5-fold increase of [(14)C]isopentenyl diphosphate incorporation into farnesyl diphosphate. Analysis of the stereochemistry revealed that all of the overexpressed farnesyl diphosphate was in the omega,E, Z-configuration. This is the first description of a short chain isoprenyl diphosphate synthase that generates products with Z-stereochemistry. Previously, all known short chain isoprenyl diphosphate synthases catalyze the synthesis of products with E-stereochemistry.
我们报道了来自结核分枝杆菌的两种独特的Z-异戊二烯基二磷酸合酶同源物的克隆、过表达及部分特性分析。第一种酶Rv1086将一个异戊二烯单元添加到ω,E-香叶基二磷酸上。产物ω,E,Z-法尼基二磷酸是第二种酶Rv2361c的假定底物。该酶再将另外七个异戊二烯单元添加到ω,E,Z-法尼基二磷酸上并释放出癸异戊烯基二磷酸。两个开放阅读框均从结核分枝杆菌H37Rv基因组中克隆出来，并在耻垢分枝杆菌中过表达。在存在各种烯丙基异戊二烯基二磷酸受体的情况下，对野生型及两种重组菌株的膜和胞质组分进行了[(14)C]异戊烯基二磷酸掺入异戊二烯基二磷酸的检测。用法尼基二磷酸孵育过表达Rv2361c的重组细胞的膜组分，结果显示[(14)C]异戊烯基二磷酸掺入癸异戊烯基二磷酸的量增加了10倍。用香叶基二磷酸孵育过表达Rv1086的重组细胞的膜组分，结果显示[(14)C]异戊烯基二磷酸掺入法尼基二磷酸的量增加了5倍。立体化学分析表明，所有过表达的法尼基二磷酸均为ω,E,Z-构型。这是首次描述生成具有Z-立体化学产物的短链异戊二烯基二磷酸合酶。此前，所有已知的短链异戊二烯基二磷酸合酶均催化生成具有E-立体化学产物的合成反应。
