Specificity and Activity of Fungal Polyketide-Nonribosomal Peptide Synthases

氰基结合蛋白是核糖体产生的、翻译后修饰的多肽(RIPP)。他们的 功能尚不清楚，但其中一些很可能在生产的共生关系中很重要 蓝藻与宿主动物有一定的微摩尔毒性。在自然界中，高度保守 氰基肌动蛋白途径产生许多不同的产物，到目前为止，每一种途径都是在实验室研究的 具有处理差异很大的多肽的能力。这项研究的目标是推动 对多种氰基丁酸中最终修饰酶底物选择性的认识 并推进旨在制造新的蓝藻毒素的生物工程努力。浩瀚无边 每条途径的产物多样性和某些氰基丁酸酶的已知活性 天然产物使氰基肌动蛋白途径成为产生新药先导的一个有吸引力的系统。 氰基肌动蛋白的生物合成途径由前体多肽和修饰酶组成。这个 前体多肽包含酶识别序列和核心序列，这些序列成为 最终产品。修饰酶进行翻译后的剪裁反应，如 蛋白质分解，单个氨基酸和整个多肽的环化，氧化，甲基化 和戊烯基化反应。在广泛的产物可变性的背后是途径中的每一种酶的能力 接受核心氨基酸序列差异很大的底物。 我的具体目标是1)测试酶和底物残留物在底物选择性中的作用 氰基丁烯基转移酶，2)研究特定生长通过的机制(S) 在大肠杆菌中提高异源氰基肌动蛋白产量的条件和3)表达新的 大肠杆菌中的氰基结合蛋白，包括已知具有活性的氰基结合蛋白的变体，用于结构- 活动关系研究。实验方法包括对纯化的 在另一个实验室进行的X射线结晶学研究的支持下， 在大肠杆菌中表达的工程途径、各种表达条件的测试和纯化 新的氰基丁酸钙产品。

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.