Unique Isoprenoid Biosynthesis Machinery of the Hyperthermophilic Archaea

类异戊二烯是一个大的、多样的和重要的分子类别，包括超过23，000个已知成员。 包括甾醇、长萜醇、类胡萝卜素、倍半萜和醌。生物衍生的类异戊二烯形成了 广泛的重要健康相关产品，包括抗肿瘤剂（紫杉醇）、抗菌剂 （ipomeamarone和capsidiol）和食品色素（虾青素）。然而，由于只有低水平的类异戊二烯或其 在细菌和真核生物中，生物功能通常需要长时间缓慢积累， 相应的酶机制处于低浓度和/或特征在于低周转数。的 古生菌必然具有更高通量的类异戊二烯生物合成机制，因为它们的膜脂是基于 仅在与多元醇（通常甘油）基头基缩合的支链饱和烃上。最 通常，脂烃是C2植烷基链;并且它们通过醚与甘油基部分偶联 与细菌和真核生物的不太稳定的酯连接的膜脂质相反。许多古生菌，尤其是 超嗜热菌还含有由两端与多元醇偶联的C4植烷基链组成的类异戊二烯脂质 得到大环双极性四醚脂质。古细菌类异戊二烯醚连接磷脂的生物化学 典型地基于二植烷基甘油二醚和二联植烷基二甘油四醚的膜是本领域所特有的。 古细菌结构域;和四醚脂质生物合成的后面的步骤需要前所未有的类异戊二烯生物化学。的 类异戊二烯醚脂质的化学稳定性及其形成更稳定的脂质膜结构的能力， 导致这些分子种类的许多重要的新兴应用， 膜结合蛋白系统的递送和重构，以及用于生物传感器应用的平面脂质膜。 古细菌合成醚脂代谢途径的阐明及其新酶的表征 因此，本发明将为合成工业上有用的类异戊二烯醚脂质和 扩大了可获得的类异戊二烯化学用于广泛的商业应用。该项目的重点是 一种极端嗜热、异养硫酸盐还原古菌的类异戊二烯生物合成机制， 闪烁古生球菌，其基因组已被测序。该项目需要新的，协调的应用， 生物化学研究技术，即，在不同条件下的极端嗜热菌培养，蛋白质纯化和 酶测定（包括使用放射性标记和LC/MS）、全基因组DMA微阵列和基因克隆， 表达，（1）鉴定或确认假设的酶活性，（2）将DNA序列分配给这些酶， 酶，（3）研究其动力学和调节，（4）深入了解起源的代谢能力， 生物体，和（5）探索所发现的新型酶的合成效用。 该项目将揭示合成一类复杂分子的新生物途径， 抗癌和抗微生物药物。这些分子也可能被证明在设计新的药物输送方面是有用的 车辆和用于健康监测的生物传感器。

The isoprenoids constitute a large, diverse and important class of molecules that includes over 23,000 known members ncluding sterols, dolichols, carotenoids, sesquiterpenes, and quinones. Biologically derived isoprenoids form the basis of a broad spectrum of important health-related products including antitumor agents (taxol), antimicrobial agents (ipomeamarone and capsidiol), and food pigments (astaxanthin). However, since only low levels of isoprenoids or their slow accumulation over long periods typically are required for biological function in bacteria and eucarya, the corresponding enzymatic machinery is at low concentration and/or is characterized by low turnover number. The archaea necessarily have higher throughput isoprenoid biosynthesis machinery; as their membrane lipids are based solely on branched-chain, saturated hydrocarbons condensed with polyol (usually glycerol)-based headgroups. Most commonly, the lipid hydrocarbons are C2ophytanyl chains; and they are coupled to a glyceryl moiety through ether linkages as opposed to the less stable ester-linked membrane lipids of bacteria and eucarya. Many archaea, especially the hyperthermophiles, also contain isoprenoid lipids consisting of C4obiphytanyl chains coupled at both ends to polyols to give macrocyclic, bipolar tetraether lipids. The biochemistry of the isoprenoid ether-linked phospholipids of archaeal membranes, based typically on diphytanylglycerol diethers and dibiphytanyldiglycerol tetraethers, is unique to the archaeal domain; and the latter steps in tetraether-lipid biosynthesis entail unprecedented isoprenoid biochemistry. The chemical robustness of isoprenoid ether lipids as well as their ability to form more stable lipid membrane structures has led to a number of important emerging applications for these molecular species including usage in liposomes for drug delivery and reconstitution of membrane-bound protein systems, and in planar lipid films for biosensor applications. Elucidation of archaeal metabolic pathways for ether lipid synthesis and characterization of their novel enzymes therefore will provide new biosyntnetic tools for the synthesis of industrially useful isoprenoid ether lipids and for the expansion of accessible isoprenoid chemistry for wide-ranging commercial application. This project focuses on the isoprenoid biosynthetic machinery of an extremely thermophilic, heterotrophic sulfate-reducing archaeon, Archaeoglobus fulgidus, whose genome has been sequenced. The project entails the novel, coordinated application of biochemical research technology, i.e., extreme thermophile culture under varied conditions, protein purification and enzyme assays (including use of radiolabeling and of LC/MS), full-genome DMAmicroarrays, and gene cloning and expression, (1) to identify or to confirm hypothesized enzyme activities, (2) to assign DNA sequences to these enzymes, (3) to study their kinetics and regulation, (4) to gain insight into the metabolic capabilities of the originating organism, and (5) to explore the synthetic utility of the novel enzymes discovered. This project will uncover new biological routes for the synthesis of a complex class of molecules that includes potent anti-cancer and anti-microbial drugs. These molecules also may prove useful in the design of new drug delivery vehicles and of biosensors for health monitoring.