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.

项目概要/摘要该提案的总体目标是评估 L,L-二氨基庚二酸酯的重要性迫切需要致病菌中的转氨酶来促进抗生素的开发。开发新型抗生素以应对抗生素耐药性数量的急剧上升阻碍抗生素发展的瓶颈之一是新型细菌的鉴定。 PI 最近鉴定并阐明了二氨基庚二酸/赖氨酸的一种新变体。通过鉴定和表征 L,L-二氨基庚二酸转氨酶 In 的生物合成途径。病原菌，如沙眼衣原体，二氨基庚二酸转氨酶催化特定的二氨基彼拉酸/赖氨酸合成代谢途径中的反应对于细胞壁肽聚糖和动物，特别是人类的基因组不包含氨基酸蛋白质合成。促进二氨基彼酸/赖氨酸合成所必需的机械，因此，其中的酶。途径是新型抗生素的有吸引力的目标。我们保留了对致病菌中二氨基彼拉转氨酶的抑制作用细菌会通过抑制肽聚糖的合成而产生杀菌作用这是因为 1) 中间体内消旋二氨基庚二酸/赖氨酸起到交联作用。细菌肽聚糖中的氨基酸；2) 赖氨酸是 20 种常见蛋白氨基酸之一。拟议的研究意义重大，因为我们将评估革兰氏阴性菌中 dapL 基因的重要性。阴性细菌刺疣微菌 (Verrucomicrobium spinosum)，是致病菌衣原体最近的自由生活近亲选择性传播疾病“棘衣原体”中的细菌作为模型是因为它。采用二氨基彼拉特转氨酶途径作为肽聚糖和赖氨酸的唯一途径该生物体不具有致病性，并且可以对其进行基因操作以测试二氨基庚二酸。转氨酶是开发新型抗生素的可行靶点，我们确定了三个目标 1)。我们将使用棘弧菌作为评估二氨基彼拉特转氨酶在真细菌中的重要性使用使用转座子和/或 dapL 基因的基因替换的诱变实验的模型2)。 PI 实验室的最新研究发现，拮抗化合物会导致二氨基庚二酸的产生因此，我们将使用体外测定来辨别这些化合物的特异性。体内系统，我们将使用 V. spinosum 野生型和二氨基彼拉特转氨酶突变体来评估如果这些已鉴定的化合物对二氨基庚二酸转氨酶具有特异性，并且 3) 的最终目标项目将通过孵化来鉴定参与拮抗化合物结合的氨基酸将酶与这些化合物结合，然后通过 X 射线晶体学进行结构分析，将支持第二代抑制剂的开发。

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

DOI：
10.1007/s00216-020-02621-9
发表时间：
2020-04-22
期刊：
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
影响因子：
4.3
作者：
De Pena, Adriana Coll;Miller, Abbi;Lapizco-Encinas, Blanca H.
通讯作者：
Lapizco-Encinas, Blanca H.

Aeromonas hydrophila RIT668 and Citrobacter portucalensis RIT669-Potential Zoonotic Pathogens Isolated from Spotted Turtles.

DOI：
10.3390/microorganisms8111805
发表时间：
2020-11-17
期刊：
Microorganisms
影响因子：
4.5
作者：
Thomas SG;Abajorga M;Glover MA;Wengert PC;Parthasarathy A;Savka MA;Wadsworth CB;Shipman PA;Hudson AO
通讯作者：
Hudson AO