类异戊二烯生物合成非甲羟戊酸途径中一种必需酶GcpE的功能特性
Functional characterization of GcpE, an essential enzyme of the non-mevalonate pathway of isoprenoid biosynthesis.
作者信息
Kollas Ann-Kristin, Duin Evert C, Eberl Matthias, Altincicek Boran, Hintz Martin, Reichenberg Armin, Henschker Dajana, Henne Anke, Steinbrecher Irina, Ostrovsky Dmitry N, Hedderich Reiner, Beck Ewald, Jomaa Hassan, Wiesner Jochen
机构信息
Biochemisches Institut, Justus-Liebig-Universität Giessen, Friedrichstrasse 24, 35392, Giessen, Germany.
出版信息
FEBS Lett. 2002 Dec 18;532(3):432-6. doi: 10.1016/s0014-5793(02)03725-0.
The gcpE gene product controls one of the terminal steps of isoprenoid biosynthesis via the mevalonate independent 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. This pathway is utilized by a variety of eubacteria, the plastids of algae and higher plants, and the plastid-like organelle of malaria parasites. Recombinant GcpE protein from the hyperthermophilic bacterium Thermus thermophilus was produced in Escherichia coli and purified under dioxygen-free conditions. The protein was enzymatically active in converting 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the presence of dithionite as reductant. The maximal specific activity was 0.6 micromol x min(-1) x mg(-1) at pH 7.5 and 55 degrees C. The kcat value was 0.4 s(-1) and the K(m) value for HMBPP 0.42 mM.
gcpE基因产物通过甲羟戊酸非依赖型2-C-甲基-D-赤藓糖醇-4-磷酸（MEP）途径控制类异戊二烯生物合成的一个终端步骤。多种真细菌、藻类和高等植物的质体以及疟原虫的类质体细胞器都利用该途径。嗜热栖热菌的重组GcpE蛋白在大肠杆菌中产生，并在无氧条件下纯化。该蛋白在连二亚硫酸盐作为还原剂存在的情况下，具有将2-C-甲基-D-赤藓糖醇-2,4-环二磷酸（MEcPP）转化为（E）-4-羟基-3-甲基-丁-2-烯基二磷酸（HMBPP）的酶活性。在pH 7.5和55℃时，最大比活性为0.6微摩尔×分钟-1×毫克-1。kcat值为0.4秒-1，HMBPP的Km值为0.42毫摩尔。
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