Biosynthesis of Mycotoxin Fumonisins: Characterization of Enzymes for Vicinal Diol and Tricarballylic Ester Formation

伏马菌素是由几种真菌病原体产生的小分子毒素，包括黄萎病镰刀菌。这种真菌是玉米和玉米衍生产品中广泛存在的污染物。尽管伏马菌素很重要，但这些毒素产生的生化和分子基础在很大程度上仍不清楚。该研究的总体目标是了解单个生物合成步骤的分子机制细节，这些步骤导致伏马毒素在黄萎病螺旋体中形成。本项目的目标是表征两组酶，它们在伏马菌素的碳骨架上引入两种不寻常的功能，邻二醇和三羧酸酯。这些官能团的引入对真菌毒素的成熟至关重要，没有这些官能团，代谢物就没有生物活性。该项目设定了两个目标：(1)表达和表征直接在毒素的线性碳链上引入邻二醇的氧化酶；(2)确定由一种独特的非核糖体肽合成酶复合物催化的化学反应，该酶将二醇转化为两种三羧酸酯。通过两个相邻碳的直接氧化来引入相邻二醇代表了碳链功能化的新方法，而由络合物催化的酯键形成是非核糖体肽合成酶催化碳-氧键而不是典型的碳-氮键形成的第一个例子。这项研究有几个重要的更广泛的影响。首先，研究生和本科生将在天然产物生物合成领域接受广泛的培训。此外，从该项目中获得的基于研究的知识将丰富和加强内布拉斯加大学林肯分校天然产物生物合成新课程的发展。此外，该项目将促进合成化学家和生物化学家之间的强有力合作，这将有助于提供有关致病真菌利用的广泛机制的关键知识，并有助于提高公众对真菌毒素的认识。这在像内布拉斯加州这样的农业州尤其重要，内布拉斯加州是美国主要的玉米产地之一。

Fumonisins are small molecular toxins, which are produced by several fungal pathogens, including Fusarium verticillioides. This fungus is a widespread contaminant of corn and maize-derived products. Despite the importance of fumonisins, the biochemical and molecular basis for the production of these toxins remains largely uncharacterized. The overall goal of the research is to understand the molecular mechanistic details of the individual biosynthetic steps that lead to the formation of fumonisins in F. verticillioides. The goal of this project is to characterize two groups of enzymes that introduce two unusual functionalities, a vicinal diol and tricarballylic esters, on the carbon backbone of fumonisins. The introduction of these functional groups is crucial for the maturation of mycotoxins, without which the metabolites are not biologically active. Two objectives are set forth for the project: (1) to express and characterize oxidation enzymes that directly introduce the vicinal diol on the linear carbon chain of the toxins and (2) to determine the chemical reactions catalyzed by a unique nonribosomal peptide synthetase complex that converts the diol into two tricarballylic esters. The introduction of a vicinal diol by the direct oxidation of two neighboring carbons represents a new approach to functionalize the carbon chain, whereas the ester bond formation catalyzed by the complex is the first example that a nonribosomal peptide synthetase catalyzes a carbon-oxygen, instead of the typical carbon-nitrogen, bond formation.The research has several significant broader impacts. First and foremost, graduate and undergraduate students will receive extensive training in the area of natural product biosynthesis. In addition, research-based knowledge obtained from the project will enrich and enhance the development of a new course in natural product biosynthesis at the University of Nebraska-Lincoln. Furthermore, the project will promote strong collaborations between a synthetic chemist and a biochemist, which will help provide critical knowledge regarding widespread mechanisms utilized by pathogenic fungi and help promote public awareness of mycotoxins. This is especially significant in an agricultural state like Nebraska, one of the nation's major corn producers.