Genetic and structural analysis of L,L-diaminopimelate aminotransferase (DapL): An attractive target for the development of narrow-spectrum antibiotics

L,L-二氨基庚二酸转氨酶 (DapL) 的遗传和结构分析：窄谱抗生素开发的一个有吸引力的靶点

基本信息

批准号：
9513721
负责人：
ANDRE O HUDSON
金额：
$ 3.08万
依托单位：
ROCHESTER INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-06 至 2020-12-31
项目状态：
已结题

项目摘要

PROJECT SUMMARY/ABSTRACT The overarching goal of this proposal is to assess the essentiality of L,L-diaminopimelate aminotransferase in pathogenic bacteria to facilitate the development of antibiotics. There is an urgent need for the development of novel antibiotics to combat the drastic rise in the number of antibiotic resistant bacteria. One of the bottlenecks that is impeding the development of antibiotics is the identification of novel enzymatic targets. The PI recently identified and elucidated a novel variant of the diaminopimelate/lysine biosynthesis pathway by identifying and characterizing the enzyme L,L-diaminopimelate aminotransferase. In pathogenic bacteria, such as Chlamydia trachomatis, diaminopimelate aminotransferase catalyzes a specific reaction in the diaminopimelate/lysine anabolic pathway that is necessary for both cell wall peptidoglycan and amino acid protein synthesis. The genomes of animals, particularly humans, do not contain the genetic machinery necessary to facilitate the synthesis of diaminopimelate/lysine. As such, the enzymes in this pathway are attractive targets for novel antibiotics. We hypothesize that inhibition of diaminopimelate aminotransferase in the pathogenic bacteria will cause a bactericidal effect through inhibition of peptidoglycan synthesis and protein synthesis. This is because 1) the intermediate meso-diaminopimelate/lysine serves a cross-linking amino acid in the peptidoglycan of bacteria and 2) lysine is one of the 20 common proteogenic amino acids. The proposed research is significant since we will assess the essentiality of the dapL gene in the Gram- negative bacterium Verrucomicrobium spinosum, the closest free living relative of Chlamydia, the causative bacterium in the sexually transmitted disease “Chlamydia”. V. spinosum was chosen as a model because it employs the diaminopimelate aminotransferase pathway as the sole pathway for peptidoglycan and lysine biosynthesis. The organism is not pathogenic and it can be genetically manipulated. To test if diaminopimelate aminotransferase is a feasible target for the development of novel antibiotics we have delineated three aims. 1) We will assess the essentiality of diaminopimelate aminotransferase in eubacteria using the V. spinosum as a model using mutagenesis experiments employing transposon and/or gene replacement of the dapL gene. 2) Recent studies from PI’s lab have identified antagonistic lead compounds towards diaminopimelate aminotransferase using in vitro assays. As such, we will discern the specificity of these compounds using a in vivo system where we will use V. spinosum wild type and diaminopimelate aminotransferase mutants to assess if these identified compounds are specific for diaminopimelate aminotransferase and 3) The final aim of the project will identify the amino acids that are involved in the binding of antagonistic compounds by incubating the enzyme with these compounds followed by structural analyses facilitated by X-ray crystallography, which will underpin the development of second generation inhibitors.

项目摘要/摘要该提案的总体目标是评估L，l-二氨基二亚匹配的本质致病细菌中的氨基转移酶，以促进抗生素的发展。迫切需要为了开发新型抗生素，以抵抗抗生素耐药性数量的急剧上升细菌。阻碍抗生素发展的瓶颈之一是新颖的识别酶促靶标。 PI最近鉴定并阐明了二氨基二二酰胺/赖氨酸的新型变体通过鉴定和表征酶L，l-二氨基二酰胺氨基转移酶的生物合成途径。致病细菌，例如沙眼衣原体，二氨基二粘酸酯氨基转移酶催化特定在细胞壁辣椒和二氨基二二二二二二二二酰胺酸盐/赖氨酸合成代谢途径中的反应氨基酸蛋白合成。动物，尤其是人类的基因组不包含遗传促进二氨基二二酰胺/赖氨酸的合成所需的机械。因此，其中的酶途径是新型抗生素的有吸引力的靶标。我们假设抑制致病性二氨基二二二二二二氨基氨基转移酶细菌将通过抑制PepperyDoglycan合成和蛋白质合成。这是因为1）中间中间二氨基二二二二胺/赖氨酸交联链接细菌的辣聚糖中的氨基酸和2）赖氨酸是20种常见的蛋白质氨基酸之一。拟议的研究很重要，因为我们将评估DAPL基因在革兰氏集中的重要性阴性细菌verrucomicrobium spinosum，是衣原体的最接近的自由生活亲戚，是病因性传播疾病“衣原体”中的细菌。 V. Spinosum被选为模型，因为它采用二氨基二键二二酰氨基转移酶途径作为辣椒和赖氨酸的唯一途径生物合成。该生物不是致病性的，可以在遗传上操纵。测试二氨基二二二二次氨基转移酶是开发新型抗生素的可行靶标，我们描述了三个目标。 1）我们将使用V. spinosum作为A作为A 使用诱变实验的模型，该实验采用了转座子和/或基因替代DAPL基因的模型。 2） PI实验室的最新研究已经确定了拮抗铅化合物对二氨基二键型的化合物氨基转移酶使用体外测定法。因此，我们将使用A中辨别这些化合物的特异性体内系统，我们将使用V.脊柱野生型和二氨基二键型氨基转移酶突变体进行评估如果这些已鉴定的化合物特异性是二氨基二二键二氨基转移酶的特异性，那么3）项目将通过孵化来鉴定拮抗化合物结合的氨基酸具有这些化合物的酶，然后是通过X射线晶体学制备的结构分析，将支持第二代抑制剂的发展。

项目成果

期刊论文数量（12）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Amino acid-derived defense metabolites from plants: A potential source to facilitate novel antimicrobial development.

DOI：
10.1016/j.jbc.2021.100438
发表时间：
2021-01
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Parthasarathy A;Borrego EJ;Savka MA;Dobson RCJ;Hudson AO
通讯作者：
Hudson AO

Structure-Function Studies of the Antibiotic Target l,l-Diaminopimelate Aminotransferase from Verrucomicrobium spinosum Reveal an Unusual Oligomeric Structure.

来自刺疣微菌 (Verrucomicrobium spinosum) 的抗生素靶点 l,l-二氨基庚二酸转氨酶的结构-功能研究揭示了一种不寻常的寡聚结构。

DOI：
10.1021/acs.biochem.0c00185
发表时间：
2020
期刊：
Biochemistry
影响因子：
2.9
作者：
Weatherhead,AnthonyW;Crowther,JenniferM;Horne,ChristopherR;Meng,Yanxiang;Coombes,David;Currie,MichaelJ;Watkin,SerenaAJ;Adams,LilyE;Parthasarathy,Anutthaman;Dobson,RenwickCJ;Hudson,AndréO
通讯作者：
Hudson,AndréO

Comparative Molecular Dynamics Simulations Provide Insight Into Antibiotic Interactions: A Case Study Using the Enzyme L,L-Diaminopimelate Aminotransferase (DapL).

比较分子动力学模拟深入了解抗生素相互作用：使用 L,L-二氨基庚二酸氨基转移酶 (DapL) 的案例研究。

DOI：
10.3389/fmolb.2020.00046
发表时间：
2020
期刊：
Frontiers in molecular biosciences
影响因子：
5
作者：
Adams,LilyE;Rynkiewicz,Patrick;Babbitt,GregoryA;Mortensen,JamieS;North,RachelA;Dobson,RenwickCJ;Hudson,AndréO
通讯作者：
Hudson,AndréO

Creation of an electrokinetic characterization library for the detection and identification of biological cells