POLYAMINE BIOSYNTHESIS AND FUNCTION

多胺，腐胺，亚精胺和精胺，是所有活细胞中的主要多元化合物。这些胺已被证明是重要的许多系统相关的生长和分化。我们感兴趣的是这些多胺是如何合成的，它们的生物合成和降解是如何调节的，它们的生理功能，它们在体内的作用方式，以及各种生物合成酶的结构特征。为此目的，我们已经构建了空突变体在大肠杆菌和酿酒酵母的生物合成步骤中的每一个，并已准备过表达系统的生物合成酶。我们目前的研究旨在扩展我们对这些胺和生物合成酶的生物化学，调节和遗传学的研究。啤酒。在本年度，我们继续研究了鸟氨酸脱羧酶的调节，该酶是多胺形成的关键酶，并且对多胺的生理功能研究特别感兴趣。在培养基中加入亚精胺后，鸟氨酸脱羧酶的水平显著降低，这是由于新合成的抑制和现有酶的降解。我们已经发现，与文献中先前的报道相反，这种降解的重要部分涉及蛋白水解活性的诱导。如果蛋白质合成受到抑制，则不会发生这种诱导。在E.大肠杆菌中，我们已经开发了大量的纯化酵母鸟氨酸脱羧酶，S-腺苷甲硫氨酸脱羧酶，亚精胺合酶和精胺合酶的晶体学和其他结构研究的分离方法。S-腺苷甲硫氨酸脱羧酶的结构是特别感兴趣的，因为它作为酶原形成，并且在酶促反应后裂解形成酶活性所必需的脱乙酰基。我们还利用PCR技术克隆了E.大肠杆菌谷胱甘肽亚精胺合酶基因作为该化合物在大肠杆菌中功能研究的初始阶段。杆菌- 腐胺，亚精胺，精胺，酵母，多胺。鸟氨酸脱羧酶

The polyamines, putrescine, spermidine, and spermine, are major polybasic compounds in all living cells. These amines have been shown to be important for many systems related to growth and differentiation. We have been interested in how these polyamines are synthesized, how their biosynthesis and degradation are regulated, their physiologic functions, how they act in vivo, and in characterizing the structure of the various biosynthetic enzymes. For this purpose we have constructed null mutants in each of the biosynthetic steps in both Escherichia coli and in Saccharomyces cerevisiae, and have prepared overexpression systems for the biosynthetic enzymes. Our present studies are directed at extending our studies on the biochemistry, regulation and genetics of these amines and of the biosynthetic enzymes in S. cerevisiae. During the current year we have continued our studies on the regulation of the enzyme ornithine decarboxylase which is a key enzyme in the formation of the polyamines, and is of special interest in studies on the physiological function of the polyamines. The ornithine decarboxylase levels are markedly lowered after the addition of spermidine to the medium, both by repression of new synthesis and by degradation of the existing enzyme. We have found that, contrary to previous reports in the literature, an important part of this degradation involves the induction of a proteolytic activity. This induction does not occur if protein synthesis is inhibited. Using overexpression systems in E. coli, we have developed methods for the isolation of large quantities of purified yeast ornithine decarboxylase, S-adenosylmethionine decarboxylase, spermidine synthase and spermine synthase for crystallographic and other structural studies. The structure of S-adenosylmethionine decarboxylase is of particular interest since it is formed as a proenzyme and is post- translationally cleaved to from a pyruvoyl group that is essential for enzymatic activity. We have also cloned (with PCR technology) the E. coli gene for glutathionylspermidine synthase as the initial phase of a study of the function of this compound in E. coli. - putrescine, spermidine, spermine, yeast, polyamines. ornithine decarboxylase, S- adenosylmethionine decarboxylase