Mutagenesis of Pyridoxal Phosphate Dependent Enzymes

磷酸吡哆醛依赖性酶的诱变

• 批准号: 6765257
• 负责人: JACK F KIRSCH
• 金额: $ 43.84万
• 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 2005-07-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6765257
• 关键词: Escherichia coli; S adenosylmethionine; bacterial genetics; bacterial proteins; binding proteins; chemical kinetics; chimeric proteins; cystathionine beta synthase; enzyme inhibitors; enzyme model; enzyme substrate analog; enzyme substrate complex; functional /structural genomics; intermolecular interaction; lyase; microorganism metabolism; model design /development; molecular site; physical model; protein engineering; protein sequence; protein structure function; pyridoxal phosphate; site directed mutagenesis; structural biology; transaminases

ACC synthase catalyzes the conversion of S- adenosylmethionine (SAM) to amino cyclopropane carboxylate (ACC). ACC is the immediate precursor to ethylene, the plant hormone responsible for, inter alia, wound healing, fruit ripening and senescence. We plan to solve X-ray structures and biochemically characterize SAM-analog complexes with wild type and mutant enzymes in order to understand the special chemistry leading to the formation of the unique cyclopropane ring. While rational enzyme design has enjoyed some success, directed evolution has opened up the possibility of discovering mutations far from the catalytic site of an enzyme that effect both catalytic rates and reaction specificity. This novel technology will be adopted to convert aspartate aminotransferase to tyrosine aminotransferase and ACC synthase to a SAM aminotransferase. The functions of two genes (yjiR and ydcR), which code for two previously unknown pyridoxal phosphate (PLP) binding proteins, will be studied by a combination of bioinformatics, biochemical and nutritional microbiology techniques. The one known SAM aminotransferase, diaminopelargonic acid (DAPA) synthase from the biotin synthetic pathway, will be investigated to try to understand how two enzymes process the same substrate to different products-ACC synthase directs SAM to ACC while DAPA synthase converts it to DAPA. Mutations in cystathionine-beta-synthase are frequently responsible for human homocystinuria. The mechanism of action of this PLP-dependent enzyme will be pursued with particular reference to try to understand how the mutations contribute to the pathology.

ACC合成酶催化S-腺苷蛋氨酸（SAM）转化为氨基环丙烷羧酸酯（ACC）。ACC是乙烯的直接前体，乙烯是负责伤口愈合、果实成熟和衰老的植物激素。我们计划求解具有野生型和突变型酶的sam -类似物的x射线结构和生化特征，以了解导致独特环丙烷环形成的特殊化学。虽然合理的酶设计已经取得了一些成功，但定向进化已经开启了发现远离酶催化位点的突变的可能性，这些突变会影响酶的催化速率和反应特异性。该技术将用于将天冬氨酸转氨酶转化为酪氨酸转氨酶，将ACC合成酶转化为SAM转氨酶。两个基因（yjiR和ydcR）编码两个以前未知的磷酸吡哆醛（PLP）结合蛋白，将通过生物信息学、生化和营养微生物学技术的结合来研究它们的功能。我们将研究已知的SAM转氨酶，即生物素合成途径中的二氨基膦酸（DAPA）合成酶，以试图了解两种酶如何将相同的底物加工成不同的产物——ACC合成酶将SAM转化为ACC，而DAPA合成酶将其转化为DAPA。胱氨酸-合成酶的突变通常是人类同型半胱氨酸尿的原因。这种plp依赖性酶的作用机制将被特别研究，以试图了解突变是如何导致病理的。