Studies of Functional Conversion of Prenyltransferase

1. 我们在产生C_<15>产物的法尼酯二磷酸合成酶中引入定点诱变来改变其产物的链长。第一个富天冬氨酸基序附近的氨基酸残基，参与底物结合，根据先前的信息被取代。因此，我们成功地改变了酶的性质，并创造了产生C_<10>产物的突变酶，香叶二磷酸。这一结果支持了我们关于戊烯基转移酶产物链长决定机理的假设。此外，由于二磷酸香叶醇是单萜烯的前体，该突变体有可能应用于工业生产。根据之前的信息，我们在七戊烯基二磷酸合成酶中引入了位点定向诱变，产生了C_<35>的产物。我们成功地将其改变为产生更短或更长的产物的突变酶。七戊烯基二磷酸合成酶根据其四级结构的不同可分为中链戊烯基转移酶。我们的研究结果表明，中链戊烯基转移酶也具有与戊烯基转移酶相同的产物链长度决定机制。作为改变酶性质的新靶点，我们克隆了几种新型戊烯基转移酶的基因，其中包括一种具有从未知道的产物特异性的基因。阐明它们的酶促机制及其性质的变化将为戊烯基转移酶的研究提供有用的知识。此外，它们也可能为我们提供酶的热稳定性信息，因为所有克隆的酶都来源于嗜热微生物，并且都是热稳定性的。

1. We introduced site-directed mutageneses in farnesyl diphosphate synthase, which yields C_<15> product, to change the chain length of its product. The amino acid residues proximate to the first aspartate rich motif, which is involved in substrate-binding, were substituted according to previous information. Consequently, we succeeded in altering the property of the enzyme and in creating the mutant enzyme which yields C_<10> product, geranyl diphosphate. The result supports our hypothesis on the mechanism of product chain length determination of prenyltransferase. Besides, the mutant might be applied in industrial use because geranyl diphosphate is the precursor of monoterpenes.2. We introduced site-directed mutageneses in heptaprenyl diphosphate synthase, which yields C_<35> product, according to previous information. We succeeded in changing it to the mutant enzymes that yield shorter or longer products. Heptaprenyl diphosphate synthase is classified into medium-chain prenyltransferase based on the difference of quaternary structure. The result of our studies revealed that medium-chain prenyltransferase also shares the mechanism of product chain length determination common to prenyltransferases.3. As new targets for changing the enzymatic properties, we cloned the genes of several novel prenyltransferases, including the one which has the product specificity that has never known. Elucidation of their enzymatic mechanism and change of their properties would give us useful knowledge for the research of prenyltransferase. Besides, they might also provide us information about the thermostability of enzymes because all of the cloned enzymes are derived from thermophilic microorganisms and are thermostable.