Schulbach M C, Mahapatra S, Macchia M, Barontini S, Papi C, Minutolo F, Bertini S, Brennan P J, Crick D C
Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523, USA.
J Biol Chem. 2001 Apr 13;276(15):11624-30. doi: 10.1074/jbc.M007168200. Epub 2001 Jan 4.
We have recently shown that open reading frame Rv1086 of the Mycobacterium tuberculosis H37Rv genome sequence encodes a unique isoprenyl diphosphate synthase. The product of this enzyme, omega,E,Z-farnesyl diphosphate, is an intermediate for the synthesis of decaprenyl phosphate, which has a central role in the biosynthesis of most features of the mycobacterial cell wall, including peptidoglycan, arabinan, linker unit galactan, and lipoarabinomannan. We have now purified Z-farnesyl diphosphate synthase to near homogeneity using a novel mycobacterial expression system. Z-Farnesyl diphosphate synthase catalyzed the addition of isopentenyl diphosphate to omega,E-geranyl diphosphate or omega,Z-neryl diphosphate yielding omega,E,Z-farnesyl diphosphate and omega,Z,Z-farnesyl diphosphate, respectively. The enzyme has an absolute requirement for a divalent cation, an optimal pH range of 7-8, and K(m) values of 124 micrometer for isopentenyl diphosphate, 38 micrometer for geranyl diphosphate, and 16 micrometer for neryl diphosphate. Inhibitors of the Z-farnesyl diphosphate synthase were designed and chemically synthesized as stable analogs of omega,E-geranyl diphosphate in which the labile diphosphate moiety was replaced with stable moieties. Studies with these compounds revealed that the active site of Z-farnesyl diphosphate synthase differs substantially from E-farnesyl diphosphate synthase from pig brain (Sus scrofa).
我们最近发现，结核分枝杆菌H37Rv基因组序列中的开放阅读框Rv1086编码一种独特的异戊二烯基二磷酸合酶。该酶的产物ω，E，Z-法呢基二磷酸是合成癸异戊烯基磷酸的中间体，癸异戊烯基磷酸在结核分枝杆菌细胞壁大多数成分的生物合成中起核心作用，这些成分包括肽聚糖、阿拉伯聚糖、连接单元半乳聚糖和脂阿拉伯甘露聚糖。我们现在使用一种新型分枝杆菌表达系统将Z-法呢基二磷酸合酶纯化至接近均一。Z-法呢基二磷酸合酶催化将异戊烯基二磷酸添加到ω，E-香叶基二磷酸或ω，Z-橙花基二磷酸上，分别生成ω，E，Z-法呢基二磷酸和ω，Z，Z-法呢基二磷酸。该酶绝对需要二价阳离子，最适pH范围为7至8，异戊烯基二磷酸的K(m)值为124微摩尔，香叶基二磷酸的K(m)值为38微摩尔，橙花基二磷酸的K(m)值为16微摩尔。设计并化学合成了Z-法呢基二磷酸合酶的抑制剂，作为ω，E-香叶基二磷酸的稳定类似物，其中不稳定的二磷酸部分被稳定部分取代。对这些化合物的研究表明，Z-法呢基二磷酸合酶的活性位点与猪脑（Sus scrofa）中的E-法呢基二磷酸合酶有很大不同。
