SITE DIRECTED MUTAGENESIS OF THE E COLI GCD GENE

描述：藜麦蛋白已成为第三大类 脱氢酶，对许多细菌的生物能量学是必不可少的 属。藜麦蛋白也存在于高等真核生物中， 可能参与必要的过程，如组织发育 以及人类、动物和植物的分化。有关以下内容的信息 与NADP相比，藜麦蛋白脱氢酶受到严重限制。 依赖脱氢酶和含黄素脱氢酶。因此，根本 这类酶的分子遗传和生化研究 必不可少的。长远目标是加强基本知识，包括 藜麦蛋白的结构和功能。具体来说，它们将使用 大肠埃希菌藜蛋白葡萄糖脱氢酶的研究 蛋白质。9个克隆的PQQ-1的氨基酸序列分析 脱氢酶揭示了一个高度保守的区域，包括 既有相同的也有相似的氨基酸。有人提议这一点 结构域在酶的结构和功能中起着至关重要的作用。 重要的是，高度保守的区域存在于可溶的 醋酸钙葡萄糖脱氢酶是一种不共享NO的酶 其他与其余8个PQQ-脱氢酶同源。他们已经使用了 丙氨酸定点突变扫描四种氨基酸 在所有九种蛋白质中都是相同的高度保守的区域。他们 已经对其中3个突变株进行了检测，结果表明酶活性是 大大减少了。他们还用氨基取代了其中的3个。 具有截然不同的R基团的酸，并获得了两个击倒和 接近淘汰赛(7%。控制)。初步数据显示， 击倒并不是折叠或折叠过程中轻微失败的结果 运送到周质。GDH的结构模型已被 在本实验室和克里斯·安东尼的实验室中使用 藜麦蛋白甲醇脱氢酶的晶体结构 由Anthony和他的同事出版。在GDH和NMH中，高度 保守区是贝塔螺旋桨折叠基序的一部分 在包括流感病毒神经氨酸酶在内的其他蛋白质中有报道。 他们希望对蛋白质的基本理解有所贡献 藜麦蛋白的结构及其独特作用机制(S) 脱氢酶。该项目的第二个具体目标是加强 阿尔弗雷德大学生物系的研究环境 并在这方面促进研究和教育的融合 以本科生为主的机构。

DESCRIPTION: Quinoproteins have emerged as the third major class of dehydrogenases and are essential to the bioenergetics of many bacterial genera. Quinoproteins are also present in higher eukaryotes and probably participate in essential processes such as tissue development and differentiation in humans, animals and plants. Information about quinoprotein dehydrogenases is severely limited compared to NADP- dependent and flavin-containing dehydrogenases. Therefore, fundamental molecular genetic and biochemical studies of this class of enzymes are essential. The long term objective is to enhance basic knowledge of quinoprotein structure and function. Specifically, they will use the E. coli quinoprotein glucose dehydrogenase to study this class of proteins. Analysis of the deduced amino acid sequences of 9 cloned PQQ- dehydrogenases has revealed a 'highly conserved' region that includes both identical and similar amino acids. It was proposed that this domain plays an essential role in enzyme structure/function. Importantly, the highly conserved region is present in the soluble glucose dehydrogenase of A. calcoaceticus , an enzyme that shares no other homology with the 8 remaining PQQ-dehydrogenases. They have used site-directed mutagenesis to alanine-scan four amino acids from the highly conserved region that are identical in all nine proteins. They have assayed 3 of these mutants and shown that enzyme activity is greatly reduced. They have also replaced 3 of these sites with amino acids with drastically different R groups and obtained two knockouts and a near knockout (7%. of control). Preliminary data show that the knockouts are not the result of a trivial failure in folding or transport to the periplasm. Structural models of GDH have been developed in this laboratory and in Chris Anthony's laboratory using the crystal structure of the quinoprotein methanol dehydrogenase (MDH) published by Anthony and coworkers. In both GDH and NMH, the highly conserved region is part of a beta-propeller fold motif that has been reported in other proteins including the influenza virus neuraminidase. They expect to contribute to the basic understanding of protein structure as well as the unique mechanism(s) of quinoprotein dehydrogenases. A second specific aim of this project is to strengthen the research environment within the Division of Biology at Alfred University and promote the integration of research and education in this predominantly undergraduate institution.