Mutagenesis of Pyridoxal Phosphate Dependent Enzymes

ACC合酶催化S-腺苷甲硫氨酸（SAM）转化为氨基环丙烷羧酸（ACC）. ACC是乙烯的直接前体，乙烯是负责伤口愈合、果实成熟和衰老等的植物激素。 我们计划解决X射线结构和生物化学表征SAM类似物复合物与野生型和突变酶，以了解特殊的化学导致独特的环丙烷环的形成。 虽然合理的酶设计已经取得了一些成功，定向进化已经开辟了发现远离酶催化位点的突变的可能性，这些突变影响催化速率和反应特异性。 将采用这种新技术将天冬氨酸转氨酶转化为酪氨酸转氨酶，将ACC合酶转化为SAM转氨酶。 将通过生物信息学、生物化学和营养微生物学技术的结合来研究两个基因（yjiR和ydcR）的功能，这两个基因编码两种以前未知的磷酸吡哆醛（PLP）结合蛋白。 本论文将研究生物素合成途径中的二氨基壬酸（达帕）合酶，即SAM氨基转移酶，以了解两种酶如何将同一底物转化为不同的产物--ACC合酶将SAM转化为ACC，而达帕合酶将SAM转化为达帕。 胱硫醚-β-合酶的突变通常是人类同型胱氨酸尿症的原因。 这种PLP依赖性酶的作用机制将被特别关注，以试图了解突变如何导致病理学。

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.