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Studies of Functional Conversion of Prenyltransferase

异戊二烯基转移酶功能转换的研究

基本信息

批准号：
11480158
负责人：
NISHINO Tokuzo
金额：
$ 2.62万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

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.

1.在法尼基二磷酸合成酶中引入定点突变，产生C_(15)&gt；产物，改变其产物的链长。根据先前的信息，替换了与底物结合有关的第一个富含天冬氨酸基序的氨基酸残基。因此，我们成功地改变了酶的性质，并创造了产生C_&lt；10&gt；产物香叶基二磷酸的突变酶。这一结果支持了我们关于戊烯基转移酶产物链长决定机制的假说。此外，由于香叶基二磷酸是单萜的前体，该突变体有可能在工业上应用。根据以前的信息，我们在七烯基二磷酸合成酶中引入了定点突变，产生了C_&lt；35&gt；产物。我们成功地将其改变为产生更短或更长产品的突变酶。根据四级结构的不同，将七烯基二磷酸合成酶分为中链戊烯基转移酶。我们的研究结果表明，中链戊烯基转移酶与戊烯基转移酶具有共同的决定产物链长的机制。作为改变酶性质的新靶点，我们克隆了几种新的戊烯基转移酶的基因，其中包括一种具有未知产物特异性的基因。阐明它们的酶机制及其性质的变化，将为我们研究异戊烯基转移酶提供有用的知识。此外，它们还可能为我们提供有关酶的热稳定性的信息，因为所有克隆的酶都来自嗜热微生物，并且都是热稳定性的。