Molecular Evolution of Pyridoxal Phoshate Enzymes

磷酸吡哆醛酶的分子进化

• 批准号: 6973947
• 负责人: JACK F KIRSCH
• 金额: $ 22.8万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6973947
• 关键词: Escherichia coli; S adenosylmethionine; X ray; binding proteins; biotechnology; chimeric proteins; cystathionine beta synthase; directed evolution; enzyme activity; enzyme model; enzyme substrate analog; enzyme substrate complex; functional /structural genomics; lactate dehydrogenases; malate dehydrogenase; molecular site; physical model; protein protein interaction; protein structure function; pyridoxal phosphate; structural biology

DESCRIPTION (provided by applicant): The major advances in understanding protein design and function arising from genomics, and computational and structural biology, have enabled lines of enquiry to enzymology, that could not have been undertaken until recently. Directed evolution or DNA shuffling will be used to attempt to convert an important oxidation enzyme of intermediary metabolism, malate dehydrogenase, to acquire the activity of a structurally related enzyme, lactate dehydrogenase, in order to compare the results of laboratory with natural evolution. The same technology will be employed, additionally, to attempt to narrow the specificity of tyrosine aminotransferase, an enzyme that reacts with aromatic and negatively charged amino acids, to an activity that engages only the latter class. Directed evolution of a plant enzyme, involved in the production of the gaseous hormone, ethylene, will be used to attempt to convert it into an aminotransferase, a very distantly related enzyme. Human tyrosinemia type 2, has been shown to be caused by a few mutations. These mutant enzymes will be characterized, both with respect to their catalytic properties and their stability under near physiological conditions, in order to define the precise relation of the molecular defect to the disease. It is widely appreciated, that a large fraction of genome sequences (perhaps 40%) has been misannotated. A test set of diverse genome-annotated aspartate and tyrosine aminotransferases, will be prepared and characterized, for substrate specificity, in order to try to discover better rules for genome annotation of enzymes. There are very few drugs that work by disrupting protein/protein interaction. A major reason is that assays are cumbersome and expensive. A mass spectrometric method is being devised, which allows rapid high throughput assays for this purpose.

描述（由申请人提供）：基因组学、计算生物学和结构生物学在理解蛋白质设计和功能方面的重大进展，使酶学研究成为可能，直到最近才开始进行。定向进化或DNA改组将被用来尝试转换一个重要的中间代谢氧化酶，苹果酸脱氢酶，以获得一个结构相关的酶，乳酸脱氢酶的活性，以比较实验室与自然进化的结果。此外，还将采用相同的技术，试图将酪氨酸氨基转移酶（一种与芳香族和带负电荷的氨基酸反应的酶）的特异性缩小到仅与后一类反应的活性。定向进化的植物酶，参与生产的气体激素，乙烯，将被用来试图将其转化为氨基转移酶，一个非常遥远的相关酶。人类酪氨酸血症2型，已被证明是由一些突变引起的。这些突变酶的特征在于它们的催化性质和它们在接近生理条件下的稳定性，以确定分子缺陷与疾病的精确关系。人们普遍认识到，大部分基因组序列（可能40%）被错误注释。一组不同的基因组注释的天冬氨酸和酪氨酸氨基转移酶，将准备和表征，底物特异性，以试图发现更好的规则基因组注释的酶。很少有药物通过破坏蛋白质/蛋白质相互作用来起作用。一个主要的原因是，测定是繁琐和昂贵的。正在设计一种质谱法，可用于快速高通量检测。