Biosynthesis of Several Oxyvinylglycine Nonproteinogenic Amino Acids Bearing Unusual Alkoxyamine Bonds

项目摘要 天然产品在制药业中起着至关重要的作用，如药物或药物化合物的先导。 这些分子通过次级新陈代谢产生，影响细菌的毒力、共生和生存 微生物。非蛋白生成的氧乙烯基甘氨酸氨基酸是一类有趣的次要氨基酸。 以乙烯氧基的存在为特征的代谢物。这个类别的成员，例如4-(2‘- 氨基乙氧基乙烯基甘氨酸(AVG)通过抑制5‘-吡哆醛-5’-甘氨酸抑制植物萌发。 磷酸(PLP)依赖的酶1-氨基环丙烷-1-羧酸(ACC)合成酶2 报道了含乙烯基醚的氧乙烯基甘氨酸非蛋白原性氨基酸的生物合成. 探索，家庭的其他成员的组成与替代的官能团仍然存在 未定。4-甲酰氨氧乙烯基甘氨酸(FVG)是一种含N-O键的氧乙烯基甘氨酸 具有双重除草和杀菌活性。3，4假单胞菌GVG生物合成基因簇 荧光菌WH6负责FVG及其相关化合物的生物合成 鸟苷氧乙烯基甘氨酸(GOVG)和氨基氧乙烯基甘氨酸(AOVG)的确切生物合成机理 仍然不清楚。5，6在这项提议中，我将确定负责形成 FVG、GOVG和AOVG。我将使用喂养实验和体外生化检测相结合的方法 测定这三种酶生物合成途径中各酶的底物和产物 非蛋白质来源的氨基酸。乙烯基烷氧基胺在氨基酸中是不寻常的，因此我将表征 负责构建N-O键并确定其化学机制的酶。这些研究将 促进类似的氧乙烯甘氨酸非蛋白生成氨基酸的生物信息学发现。此外， 对负责N-O键形成的铁依赖酶的表征将有助于它在 合成背景，以扩大氧化生物催化剂的保留范围。

Project Summary Natural products serve crucial roles in the pharmaceutical industry as drugs or leads for drug compounds. Generated through secondary metabolism, these molecules impact the virulence, symbiosis, and survival of microorganisms. The nonproteinogenic oxyvinylglycine amino acids are an interesting class of secondary metabolites characterized by the presence of a vinyl oxygen moiety. Members of this class, such as 4-(2’- aminoethoxy)vinylglycine (AVG), are known to arrest germination in plants by inhibiting the pyridoxal 5’- phosphate (PLP)-dependent enzyme 1-aminocyclopropane-1-carboxylate (ACC) synthase.2 While the biosynthesis of vinyl ether-bearing oxyvinylglycine nonproteinogenic amino acids including AVG has been explored, the formation of other members of the family with alternative functional groups remains undetermined. 4-Formylaminooxyvinylglycine (FVG), an oxyvinylglycine containing an N–O bond, was reported to have duel herbicidal and bactericidal activity.3,4 The gvg biosynthetic gene cluster in Pseudomonas fluorescens strain WH6 is responsible for the biosynthesis of FVG and the related compounds guanidinooxyvinylglycine (GOVG) and aminooxyvinylglycine (AOVG) though the exact biosynthetic mechanism remains unclear.5,6 In this proposal I will determine the biosynthetic pathways responsible for the formation of FVG, GOVG, and AOVG. I will use a combination of feeding experiments and in vitro biochemical assays to determine the substrate and product of each enzyme along the biosynthetic pathway of these three nonproteinogenic amino acids. The vinyl alkoxyamine is unusual in amino acids, therefore I will characterize the enzyme responsible for N–O bond construction and determine its chemical mechanism. These studies will facilitate the bioinformatic discovery of similar oxyvinylglycine nonproteinogenic amino acids. Moreover, characterization of the iron-dependent enzyme responsible for N–O bond formation will facilitate its use in a synthetic context to expand the repertoire of oxidative biocatalysts.