Mutagenesis of Pyridoxal Phosphate Dependent Enzymes

磷酸吡哆醛依赖性酶的诱变

• 批准号: 6604177
• 负责人: JACK F KIRSCH
• 金额: $ 42.57万
• 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 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6604177
• 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合酶催化S-腺苷硫氨酸（SAM）转化为氨基环丙烷羧酸盐（ACC）。 ACC是乙烯的直接前体，乙烯是植物激素，负责，除其他外，伤口愈合，果实成熟和衰老。 我们计划求解X射线结构，并在生物化学上表征具有野生型和突变酶的Sam-Analog复合物，以了解导致形成独特环丙烷环的特殊化学性质。 尽管有理酶设计取得了一些成功，但定向的进化开辟了发现远离酶的催化位点的突变的可能性，从而影响催化速率和反应特异性。 这项新型技术将被采用，将天冬氨酸氨基转移酶转化为酪氨酸氨基转移酶和ACC合酶转化为SAM氨基转移酶。 两种基因（YJIR和YDCR）的功能，该功能将通过生物信息学，生化和营养微生物学技术的结合来研究两个以前未知的吡多毒素磷酸（PLP）结合蛋白。 将研究一种已知的SAM氨基转移酶，来自生物素合成途径的二氨基核酸（DAPA）合酶，以尝试了解两种酶如何将相同的底物处理为不同的乘积-ACC合成酶将SAM转化为ACC ACC ACC，而DAPA合成酶将其转换为DAPA。 胱淀粉β-合酶中的突变通常导致人类同卵巢尿症。 该PLP依赖性酶的作用机理将被提及，以特别的参考，以尝试了解突变如何对病理有效。