Mutagenesis of Pyridoxal Phosphate Dependent Enzymes

乙酰胆碱合成酶催化S-腺苷甲硫氨酸(SAM)转化为氨基环丙烷羧酸盐(ACC)。ACC是乙烯的直接前体，乙烯是一种植物激素，负责伤口愈合、水果成熟和衰老。我们计划解决X射线结构并对SAM类似物与野生型和突变型酶的络合物进行生化表征，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，但定向进化打开了发现远离酶催化部位的突变的可能性，这种突变既影响催化速度，又影响反应特异性。这项新技术将被用来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。将结合生物信息学、生化和营养微生物学技术研究两个基因(yjiR和ydcR)的功能，这两个基因编码两个以前未知的吡哆醛磷酸(PLP)结合蛋白。已知的SAM氨基转移酶，生物素合成途径中的二氨基精氨酸(DAPA)合成酶，将被研究以试图理解两种酶如何将同一底物处理成不同的产物-ACC合成酶将SAM导向ACC，而DAPA合成酶将其转化为DAPA。胱硫醚-β-合酶基因突变经常导致人类同型半胱氨酸尿症。这种依赖PLP的酶的作用机制将在特别参考的情况下进行探索，以试图了解这些突变是如何导致病理的。

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.