The origins of amino acid selectivity in the homologation pathway

同源途径中氨基酸选择性的起源

基本信息

  • 批准号:
    10729967
  • 负责人:
  • 金额:
    $ 42.42万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2023
  • 资助国家:
    美国
  • 起止时间:
    2023-09-01 至 2026-08-31
  • 项目状态:
    未结题

项目摘要

PROJECT SUMMARY The objective of this proposed project is to understand the fundamental mechanisms and functions of enzymes that catalyze homologation (insertion of a methylene group) of an amino acid side chain in natural product (NP) biosynthesis. The homologation pathway is proposed to be comprised of four enzymes, one known and three novel ones, and has a narrow substrate scope. The project is focused on determining the origins of substrate specificity in the pathway for L-Phe and L-Tyr homologation in the biosynthesis of cyanobacterium NPs anabaenopeptins. The following Aims are designed to achieve this objective: Aim 1: determination of the origins of specificity (among novel enzymes, HphA, HphB, or HphCD) in the homologation pathway of L-Phe and L-Tyr and Aim 2: rational homology-based mutagenesis to alter the substrate scope of specific homologation enzyme(s) and determination of crystal structure of these enzymes. By biochemical characterization of each enzyme in Aim 1a and establishment of the substrate profile in Aim 1b, the enzyme(s) contributing to the specificity of homologation will be identified. The substrate selective enzyme(s) will be mutated to alter or expand the substrate scope of the enzyme(s) so that nonproteinogenic or other proteinogenic amino acids can be homologated in Aim 2a. These enzymes will be characterized structurally by X-ray crystallography to further guide the mutagenesis studies in Aim 2b. NPs are one of the major sources of biological probes and medicines. Modification of known bioactive compounds is a cost- and time-effective way to discover and expand the chemical diversity of bioactive compounds to alter or improve their properties. However, the complex structure of NPs makes the specific modification of the molecules by organic chemistry challenging. Therefore, combinatorial biosynthesis that uses the recombinant biosynthetic pathway to produce “unnatural” natural products is highly attractive. Homologation of amino acid moieties is a rare modification in peptide NPs, termed nonribosomal peptides (NRPs) which are one of the major targets of NP engineering due to the flexibility of their biosynthetic pathways. Since this modification is observed only in cyanobacterial and fungal species, it has great potential to derivatize NRPs produced by these and other species, which has never been performed. The knowledge gained by this project will ultimately lead to an enzymatic and genetic tool that can derivatize a variety of NPs.
项目概要 该项目的目标是了解酶的基本机制和功能 催化天然产物 (NP) 中氨基酸侧链的同系化(亚甲基的插入) 生物合成途径被认为由四种酶组成,一种是已知的,另一种是已知的。 该项目的重点是确定底物的来源。 蓝藻纳米颗粒生物合成中 L-Phe 和 L-Tyr 同源化途径的特异性 鱼腥肽旨在实现这一目标: 目标 1:确定来源。 L-Phe 和 L-Tyr 同源途径中的特异性(新型酶 HphA、HphB 或 HphCD) 目标 2:基于合理同源性的诱变,以改变特定同源性的底物范围 酶并通过每种酶的生化表征确定这些酶的晶体结构。 目标 1a 中的酶和目标 1b 中底物谱的建立,酶有助于 底物选择性酶将被突变以改变或扩展。 酶的底物范围,以便可以将非蛋白氨基酸或其他蛋白氨基酸 这些酶将通过 X 射线晶体学进行结构表征,以进一步确定目标 2a 中的同源性。 指导 Aim 2b 中的诱变研究。 NP 是生物探针和药物的主要来源之一。 已知生物活性化合物的修饰是发现和扩展生物活性化合物的一种经济有效的方法 生物活性化合物的化学多样性改变或改善了其复杂的结构。 纳米颗粒的存在使得通过有机化学对分子进行特异性修饰具有挑战性。 组合生物合成,利用重组生物合成途径生产“非天然”的天然物质 产品非常有吸引力,氨基酸部分的同源化是肽纳米颗粒的一种罕见修饰,称为肽纳米颗粒。 非核糖体肽(NRP)由于其灵活性而成为纳米粒子工程的主要目标之一 由于这种修饰仅在蓝细菌和真菌物种中观察到,因此它具有很大的作用。 衍生化这些物种和其他物种产生的 NRP 的潜力,但从未进行过。 该项目获得的知识最终将产生一种酶促和遗传工具,可以衍生出多种 NP。

项目成果

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Shogo Mori的其他文献

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