Biochemistry and Molecular Biology of Sesquiterpene Cyclase and Squalene Synthetase from Tobacco

[408152] Chappell类异戊二烯类化合物在自然界中无处不在，对生命至关重要，并介导生物体与其环境之间的许多相互作用。此外，植物是类异戊二烯的丰富来源，对药物用途很重要。然而，我们对植物类异戊二烯生物合成途径的了解是有限的。最近的研究表明，烟草细胞悬浮培养非常适合于对类异戊二烯代谢的一个重要但假定的分支点进行详细的生理生化研究。当真菌激发子添加到细胞培养物中，培养物停止生产甾醇，而是合成和分泌抗微生物倍半萜。甾醇生物合成的下降与角鲨烯合成酶活性的抑制以及倍半萜环化酶诱导的倍半萜生物合成有关。由于这两种酶位于该途径中假定的分支点，因此一种酶的诱导和另一种酶的抑制被解释为控制碳流和最终产物形成的重要调节机制。或者，专门生产特定最终产物的独立调节的同工酶阵列可能沿着表面或膜排列。无论植物细胞内可能发生哪种代谢组织，角鲨烯合成酶和倍半萜环化酶都是甾醇和倍半萜生物合成的关键酶。这项研究的长期目标是了解这两种酶的所有方面，从对它们的酶学的全面了解到如何调节相应基因的表达的细节。目前的目标是继续确定对控制环化酶和角鲨烯合成酶基因转录率重要的DNA序列和DNA结合蛋白，并利用比较分子生物学和细菌中表达的嵌合环化酶基因的评估相结合，开始环化酶反应机制的分析。这些研究结果将有助于阐明植物类异戊二烯代谢的调节机制，并将对未来利用基因工程技术操纵这一途径具有重要意义。＊＊＊

9408152 Chappell Isoprenoids are ubiquitous in nature, essential for life and mediate a number of interactions between organisms and their environment. In addition, plants are a rich source of isoprenoids important for pharmaceutical uses. Yet, our understanding of the isoprenoid biosynthetic pathway in plants is limited. Recent studies indicate that tobacco cell suspension cultures are uniquely suited for detailed physiological and biochemical studies of one important but putative branch point in isoprenoid metabolism. When fungal elicitors are added to the cell cultures, the cultures cease sterol production and instead synthesize and secrete anti-microbial sesquiterpenoids. The decline in sterol biosynthesis has been correlated with suppression of squalene synthetase enzyme activity, and the induction of sesquiterpenoid biosynthesis with the induction of a sesquiterpene cyclase. Because these two enzymes are positioned at a putative branch point in the pathway, the induction of one enzyme and the suppression of the other are interpreted as an important regulatory mechanism controlling carbon flow and hence, end product formation. Alternatively, independently regulated arrays of isozymes dedicated to the production of specific end products might be aligned along a surface or membrane. Regardless of which metabolic organization might be occurring within a plant cell, squalene synthetase and sesquiterpene cyclase are key enzymes for sterol and sesquiterpene biosynthesis. The long range goal of this research is to understand all aspects of these two enzymes, from a complete understanding of their enzymology to the fine details of how expression of the corresponding genes are regulated. The current objectives are to continue defining the DNA sequences and DNA binding proteins important for controlling the transcription rate of the cyclase and squalene synthetase genes, and to begin an analysis of the cyclase reaction mechanism using a combination of comparative molecular biology and an evaluation of chimeric cyclase genes expressed in bacteria. Results from these studies will help elucidate the mechanisms regulating isoprenoid metabolism in plants, and will have important ramifications for future manipulations of this pathway using genetic engineering technology. ***