Studies of Isoprenoid Biosynthesis in Escherichia coli-Further clarification of metabolic pathway and it's regulation mechanism

1.在大肠杆菌法尼基转移酶缺失突变体中检测到两种新的异戊二烯基转移酶活性。虽然这些酶的量很小，但它们被认为是检测到的酶的替代品。它们可以通过向缓冲溶液中加入去污剂来纯化，就像膜蛋白一样。其中一个被鉴定为八异戊二烯基二磷酸合成酶，另一个被证明可以合成法呢基二磷酸和更长的异戊二烯基二磷酸2.我们试图阐明异戊烯基转移酶决定其产物链长的机制，目的是在大肠杆菌中生产异戊烯基二磷酸。利用随机突变技术，我们成功地改变了来源于嗜热真细菌和高度嗜热古细菌的异戊烯基转移酶产物的链长，并揭示了位于异戊烯基转移酶之间高度保守的两个富含谷氨酸基序的第一个基序之前的第五位上的氨基酸残基对产物确定机制的重要性。这是第一个人工控制异戊二烯基转移酶产物链长的报道。此外，我们还比较了各种异戊烯基转移酶的重要氨基酸残基的周围区域，并根据比较结果对它们进行了诱变。因此，我们发现异戊烯基转移酶的产物决定机制可以遗传分化。

1.We detected two novel prenyltransferase activities in a farnesyl transferase null mutant of Escherichia coli. Although the amounts of these enzymes are slight, they are thought to substitute for the detected enzyme. They can be purified by adding a detergent into buffer solution, like membrane proteins. one of them was characterized as octaprenyl diphosphate synthase, and the other was shown to synthesize farnesyl diphosphate and longer prenyl diphosphates2.We tried to elucidate the mechanism by which prenyltransferases decided chain length of their products, aiming at production of prenyl diphosphates in Escherichia coli. Using random mutagenesis technique, we succeeded to change chain length of the products.of prenyltransferases derived from a thermophilic eubacteria and a high-degree thermophilic archaea, and revealed that an amino acid residue which locates on the fifth position before the first of two aspartate-rich motifs, which are highly conserved between prenyltransferases, is important for the mechanism of product detemination. They were the first reports on the artificial control of the product's chain length of prenyltransferases. Besides, we compared surrounding regions of the important amino acid residues of various prenyltransferases and mutagenized them according to the result of comparison. Therefore we found that the mechanisms of product determination of prenyltransferases can be differentiated phylogenctically.