Plant Terpene Metabolism: Monoterpene Hydroxylases and Sesquiterpene Cyclases

大多数单萜和倍半萜是由焦磷酸戊烯基酯环化成烯烃，然后由细胞色素P450依赖的羟基化引发氧化修饰而得。对两种不同物理性质和反应复杂性的倍半萜环化酶的研究将检验这样一种假设，即相同的活性部位残基在这种酶的亲电机制中发挥类似的作用。对三个密切相关的(-)-柠檬烯羟基酶的研究，这些酶在这种简单的单萜烯烃的烯丙基羟化作用中的位置不同，将检验这样一种假设，即活性部位形貌的微小变化是这些细胞色素P450酶插入氧的区域化学基础。同源蛋白将被用来产生抗体和部分氨基酸序列，并采用分子克隆和表达策略来获得完整的初级序列和更多的酶。将使用放射性修饰剂选择性地标记环化酶的活性部位半胱氨酸和组氨酸残基，并将利用耐光底物类似物来标记环化酶和羟基酶的疏水性区域。活性部位的多肽将被制备、测序、比较并定位在蛋白质上。特定残基在环化酶催化中的作用将通过使用代表不同底物结合结构域的类似物来防止化学修饰和通过定向突变来确定。柠檬烯连接的血红素导向的抑制剂，以及19F-核磁共振方法，将被用来探索细胞色素的血红素铁与柠檬烯底物之间的空间关系。这些研究将为这些新型环酶和羟基酶催化剂的性质提供新的信息，并更清楚地了解萜类天然产物的来源。萜类化合物在植物与环境的相互作用中起着重要的作用。对萜类生物化学的了解将有助于更深入地了解这些化合物在植物中的作用。除了众所周知的商业树脂精油外，正在认真考虑将这些植物代谢物作为能源密集型化学品的可再生来源的非传统用途。广泛的萜类化合物具有药理意义，许多具有农业化学用途。从这项工作中获得的新知识将有助于努力增加这些有价值的植物代谢物的产量。

Most monoterpenes and sesquiterpenes are derived by cyclization of prenyl pyrophosphates to olefins followed by oxidative modification initiated by cytochrome P450-dependent hydroxylation. Studies with two sesquiterpene cyclases, differing in physical properties and reaction complexity, will test the hypothesis that the same active site residues play similar roles in the electrophilic mechanism of this enzyme type. Studies with three closely-related (-)-limonene hydroxylases, that differ in position of allylic hydroxylation of this simple monoterpene olefin, will test the proposal that minor alterations in active site topography underlie regiochemistry of oxygen insertion by these cytochrome P450 enzymes. Homogeneous proteins will be used to generate antibodies and partial amino acid sequences, and a molecular cloning and expression strategy adopted to obtain complete primary sequences and larger amounts of the enzymes. Radioactive modifying reagents will be employed to selectively label active site cysteine and histidine residues of the cyclases, and photolabile substrate analogs will be utilized to label hydrophobic regions of both cyclase and hydroxylase sites. Active site peptides will be prepared, sequenced, compared, and located on the proteins. The role of specific residues in cyclase catalysis will be determined by protection against chemical modification using analogs representing different substrate binding domains, and by directed mutagenesis. Limonene-tethered heme- directed inhibitors, as well as 19F-NMR methods, will be used to probe the spatial relationship between the heme iron of the cytochromes and the limonene substrate. These studies will provide new information on the nature of these novel cyclase and hydroxylase catalysts and a clearer understanding of the origin of terpenoid natural products. Terpenoids play an important role in the interaction of the plant with its environment. An understanding of terpenoid biochemistry will allow a deeper understanding of the role of these compounds in plants. In addition to the well known essential oils of resins of commerce, serious consideration is being given to nontraditional uses of these plant metabolites as renewable sources of energy- intensive chemicals. A broad range of terpenoids are of pharmacological significance and many are of agrochemical utility. New knowledge gained from this work will assist in efforts to increase the production of these valuable plant metabolites.//