SITE DIRECTED MUTAGENESIS OF THE E COLI GCD GENE

描述：藜蛋白已成为第三大类 它是许多细菌的生物能量学所必需的 属 奎宁蛋白也存在于高等真核生物中， 可能参与了组织发育等重要过程 以及人类、动物和植物的分化。 信息 与NADP相比，醌蛋白激酶受到严重限制。 依赖性和含黄素的糖苷酶。 因此，基本 这类酶的分子遗传学和生物化学研究是 具有本质意义 长期目标是提高对以下方面的基本认识： quinoprotein结构与功能 具体而言，他们将使用 E. coli quinoprotein glucose dehydrogenase来研究这类 proteins. 对9个克隆的PQQ- 酶已经揭示了一个“高度保守”的区域，包括 相同和相似的氨基酸。 有人提议， 结构域在酶的结构/功能中起着至关重要的作用。 重要的是，该高度保守的区域存在于可溶性 葡萄糖脱氢酶A.醋酸钙，一种酶， 与其余8种PQQ-内切酶具有其它同源性。 他们利用 定点突变丙氨酸扫描四个氨基酸从 在所有九种蛋白质中是相同的高度保守区域。 他们 已经测定了这些突变体中的3种，并表明酶活性是 大大减少。 他们还用氨基取代了其中3个位点 具有显著不同的R基团的酸，并获得了两个敲除， 几乎击倒对手（7%， 控制）。 初步数据显示， 击倒不是折叠中的一个微不足道的失败的结果， 运输到周质。 GDH的结构模型已被 在这个实验室和克里斯·安东尼的实验室里， 醌蛋白甲醇脱氢酶（MDH）的晶体结构 安东尼和他的同事们。 在GDH和NMH中， 保守区是β-螺旋桨折叠基序的一部分， 在其他蛋白质中也有报道，包括流感病毒神经氨酸酶。 他们希望能对蛋白质的基本认识做出贡献 结构以及quinoprotein的独特机制 - 是的 该项目的第二个具体目标是加强 阿尔弗雷德生物学系的研究环境 大学，促进研究与教育的融合在此 主要是本科院校。

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.