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

• 批准号: 9171013
• 负责人: ANDRE O HUDSON
• 金额: $ 43.7万
• 依托单位: ROCHESTER INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/9171013
• 关键词: Affect; Amino Acids; Anabolism; Animals; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Genome; Binding; Cell Wall; Centers for Disease Control and Prevention (U.S.); Cessation of life; Chlamydia; Chlamydia trachomatis; Complement; Data; Development; Drug resistance in tuberculosis; Enzymes; Essential Genes; Eubacterium; Foundations; Future; Generations; Genes; Genetic; Genome; Genotype; Goals; Gram-Negative Bacteria; Health; Human; Human Genome; In Vitro; Incubated; Lead; Life; Lysine; Lysine Biosynthesis Pathway; Mediating; Methods; Modeling; Morbidity - disease rate; Multi-Drug Resistance; Mutagenesis; Mutation; Organism; Outcome; Pathway interactions; Patients; Peptidoglycan; Phenotype; Protein Biosynthesis; Pseudomonas aeruginosa; Reaction; Research; Resistance; Role; Sexually Transmitted Diseases; Specificity; Staphylococcus aureus; System; Testing; Transaminases; Vancomycin; Variant; Work; X-Ray Crystallography; aminoacid biosynthesis; analytical ultracentrifugation; bacterial resistance; bactericide; base; biophysical properties; biophysical techniques; combat; crosslink; disorder prevention; dosage; gene replacement; human mortality; in vitro Assay; in vivo; inhibitor/antagonist; interest; methicillin resistant Staphylococcus aureus; mortality; mutant; novel; pathogenic bacteria; research study; resistant strain

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.

项目总结/摘要 本提案的总体目标是评估L，L-二氨基庚二酸盐的重要性 致病菌的转氨酶，以促进抗生素的发展。迫切需要 用于开发新型抗生素，以对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抗生素的鉴定。 酶靶点PI最近鉴定并阐明了二氨基庚二酸/赖氨酸的新变体 通过鉴定和表征酶L，L-二氨基庚二酸氨基转移酶来研究生物合成途径。在 病原菌，如沙眼衣原体，二氨基庚二酸氨基转移酶催化特异性 二氨基庚二酸/赖氨酸合成代谢途径中的反应，这是细胞壁肽聚糖和 氨基酸蛋白质合成动物的基因组，特别是人类，不包含遗传基因。 促进二氨基庚二酸/赖氨酸合成所必需的机械。因此，这种酶 是新型抗生素的有吸引力的靶标。 我们推测，在致病性大肠杆菌中抑制二氨基庚二酸转氨酶， 细菌将通过抑制肽聚糖合成而产生杀菌作用， 蛋白质合成这是因为1）中间体内消旋-二氨基庚二酸/赖氨酸起到交联的作用， 赖氨酸是20种常见的蛋白质氨基酸之一。 这项研究意义重大，因为我们将评估dapL基因在革兰氏阴性杆菌中的重要性， 阴性细菌刺状疣微菌（Verrucomicrobium spinosum）是衣原体的最接近的自由生活亲属， 性传播疾病中的细菌“衣原体”。V.spinosum被选为模型，因为它 采用二氨基庚二酸转氨酶途径作为肽聚糖和赖氨酸的唯一途径 生物合成该生物体不是致病性的，它可以被基因操纵。检测二氨基庚二酸 氨基转移酶是开发新型抗生素的可行靶点，我们已经阐明了三个目标。第一章 我们将使用刺孢弧菌作为真细菌中二氨基庚二酸氨基转移酶的重要性。 使用采用转座子和/或dapL基因的基因置换的诱变实验的模型。（二） PI实验室最近的研究已经确定了对二氨基庚二酸的拮抗先导化合物 使用体外测定法测定氨基转移酶。因此，我们将使用一种新的方法来识别这些化合物的特异性。 体内系统，其中我们将使用刺葡萄球菌野生型和二氨基庚二酸转氨酶突变体来评估 如果这些鉴定的化合物对二氨基庚二酸氨基转移酶具有特异性，以及3） 该项目将通过孵育来识别参与拮抗化合物结合的氨基酸， 酶与这些化合物，随后通过X射线晶体学进行结构分析， 将成为第二代抑制剂的基础。