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.

项目摘要/摘要 这项建议的首要目标是评估L、L-二氨基苯丙酸二酯的重要性 病原菌中的转氨酶有助于抗生素的开发。有一个迫切的需要 用于开发新的抗生素，以对抗抗生素耐药性的急剧上升 细菌。阻碍抗生素发展的瓶颈之一是对新型抗生素的鉴定 酶靶标。PI最近鉴定并阐明了二氨基戊二酸/赖氨酸的一个新变体 通过鉴定和鉴定L、L-二氨基戊二酸氨基转移酶的生物合成途径。在……里面 病原菌，如沙眼衣原体，二氨基戊二酸转氨酶催化特定的 细胞壁肽聚糖和赖氨酸合成代谢途径中的反应 氨基酸蛋白质合成。动物的基因组，特别是人类的基因组，不包含基因 促进合成二胺基戊二酸/赖氨酸所需的机械。因此，这里面的酶 途径是新抗生素的诱人靶点。 我们推测，抑制二氨基戊二酸转氨酶在致病 细菌会通过抑制肽聚糖的合成而产生杀菌作用 蛋白质合成。这是因为1)中间体中位二胺/赖氨酸起交联剂的作用。 细菌肽聚糖中的氨基酸和2)赖氨酸是20种常见的蛋白生成氨基酸之一。 这项拟议的研究具有重要意义，因为我们将评估dapL基因在Gram-Gram中的重要性。 引起衣原体的最接近的自由生活亲缘的负性细菌刺毛韦氏杆菌 性传播疾病“衣原体”中的细菌。被选为模型是因为它 利用二氨基甲酸氨基转移酶途径作为肽聚糖和赖氨酸的唯一途径 生物合成。这种生物体不是致病的，它可以被基因操纵。来测试二氨基苯丙酸酯是否 转氨酶是开发新型抗生素的一个可行靶点，我们提出了三个目标。1) 我们将以棘假单胞菌为研究对象，评估真细菌中二氨基戊二酸转氨酶的重要性。 使用转座子和/或dapL基因替换的突变实验的模型。2) 最近来自Pi实验室的研究已经确定了对二氨基匹林具有拮抗作用的先导化合物 转氨酶的体外测定。因此，我们将使用in识别这些化合物的特异性 在活体系统中，我们将使用棘假单胞菌野生型和二氨基丙二酸氨基转移酶突变体来评估 如果这些已鉴定的化合物是二氨基戊二酸转氨酶的专一性化合物和3)最终目标是 该项目将通过培养鉴定参与拮抗化合物结合的氨基酸。 与这些化合物的酶随后通过X射线结晶学进行结构分析，这 将为第二代抑制剂的开发奠定基础。