Specificity and Activity of Fungal Polyketide-Nonribosomal Peptide Synthases

蓝藻毒素是核糖体产生的和后修饰的肽（RIPPs）。他们的 功能尚不清楚，但有些可能是重要的共生关系，生产 蓝藻与宿主动物有密切关系，有的具有微摩尔毒性。在自然界中， 氰氧化菌素途径产生许多不同的产物，到目前为止，每种途径都在实验室中进行了研究。 具有加工多种肽的能力。这项研究的目的是推动 对许多氰基菌素中最终修饰酶的底物选择性的理解 途径，并推进生物工程的努力，旨在使新的蓝藻。绝 每种途径的产物多样性和一些氰基菌素的已知活性 天然产物使氰基菌素途径成为生产新药先导的有吸引力的系统。 蓝藻菌素的生物合成途径包括前体肽和修饰酶。的 前体肽含有酶识别序列和核心序列， 最终产品。修饰酶进行翻译后剪裁反应，例如 蛋白水解、单个氨基酸和整个肽的环化、氧化、甲基化 和异戊烯化。在广泛的产物变异性的基础上，是途径中每种酶的能力 以接受在其核心中具有广泛不同的氨基酸序列的底物。 我的具体目标是1）测试酶和底物残基在底物选择性中的作用， 氰基菌素异戊二烯基转移酶，2）研究特定生长的机制 条件增加异源氰菌素在E. 3）表达新的 蓝藻毒素E.大肠杆菌，包括已知具有活性的蓝藻菌素的变体，用于结构- 活动关系研究。实验方法涉及对纯化的 异戊烯基转移酶，得到了另一个实验室正在进行的X射线晶体学研究的支持， 工程化途径在E.大肠杆菌，测试各种表达条件，并纯化 新的氰氧化菌素产品。

Cyanobactins are ribosomally produced and post-translationally modified peptides (RiPPs). Their functions are not known, but some are likely to be important in symbiotic relationships of producing cyanobacteria with host animals, and some have micromolar toxicity. In nature, highly conserved cyanobactin pathways produce many different products, and each pathway studied in the lab so far has the ability to process widely varying peptides. The goal of this research is to advance understanding of the substrate selectivity of the final modifying enzyme in many cyanobactin pathways, and to advance bioengineering efforts aimed at making new cyanobactins. The vast product diversity available from each pathway and the known activities of some cyanobactin natural products makes cyanobactin pathways an attractive system for producing new drug leads. Cyanobactin biosynthetic pathways consist of precursor peptides and modifying enzymes. The precursor peptides contain enzyme recognition sequences and core sequences that become the final products. The modifying enzymes carry out post-translational tailoring reactions such as proteolysis, cyclization of individual amino acids and of the whole peptide, oxidations, methylation and prenylation. Underlying the wide product variability is the ability of every enzyme in a pathway to accept substrates with widely differing amino acid sequences in their cores. My specific aims are 1) to test the role of enzyme and substrate residues in substrate selectivity of cyanobactin prenyltransferases, 2) to study the mechanism(s) through which particular growth conditions increase heterologous cyanobactin production in E. coli and 3) to express new cyanobactins in E. coli, including variants on cyanobactins known to be active, for use in structure- activity relationship studies. The experimental approach involves biochemical work on purified prenyltransferases, supported by x-ray crystallography studies being done in another lab, engineering pathways for expression in E. coli, testing various expression conditions, and purifying new cyanobactin products.