The origins of amino acid selectivity in the homologation pathway

项目总结 这个拟议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(插入亚甲基) 生物合成。同源途径被认为由四种酶组成，一种已知，三种。 新颖的，并且底物范围很窄。该项目的重点是确定底物的来源。 L-Phe和L-Tyr同源途径在蓝藻纳米粒合成中的特异性 鱼腥草素。为实现这一目标设计了以下目标：目标1：确定来源 在L-Phe和L-Tyr的同源途径中(在新的酶HphA、HphB或HphCD中)的特异性 和目标2：基于合理同源的突变以改变特定同源的底物范围 酶(S)和这些酶的晶体结构的测定。通过对每种生物化学特性的研究 Aim 1a中的酶和Aim 1b中底物谱的建立，该酶(S)对 同源的特异性将被鉴定。底物选择酶(S)将发生突变以改变或扩展 酶的底物范围(S)，以便非蛋白生成或其他蛋白生成的氨基酸可以 在Aim 2a中同源。这些酶将通过X射线结晶学进行结构表征，以进一步 指导AIM 2b中的诱变研究。核动力源是生物探针和药物的主要来源之一。 对已知的生物活性化合物进行修饰是一种既经济又省时的方法来发现和扩大 化学多样性的生物活性化合物，以改变或改善其性质。然而，复杂的结构 纳米粒子的出现，使得有机化学对其进行特定的修饰具有挑战性。因此， 利用重组生物合成途径产生“非天然”天然产物的组合生物合成 产品极具吸引力。氨基酸部分的同源是多肽NPs中罕见的修饰，称为 非核糖体肽(NRPs)由于其自身的灵活性而成为NP工程的主要靶点之一 生物合成途径。由于这种修饰只在蓝藻和真菌物种中观察到，所以它有很大的 有可能衍生这些物种和其他物种产生的NRPs，这是从未进行过的。这个 通过这个项目获得的知识将最终导致一种酶和遗传工具，可以衍生出各种 NPs的。

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.