{"title":"探索二苯甲酮类腺苷酸酶光亲和探针的最佳光亲和连接物。","authors":"Sho Konno , Fumihiro Ishikawa , Hideaki Kakeya , Genzoh Tanabe","doi":"10.1016/j.bmc.2024.117815","DOIUrl":null,"url":null,"abstract":"<div><p>The adenylation (A) domain of non-ribosomal peptide synthetases (NRPSs) catalyzes the adenylation reaction with substrate amino acids and ATP. Leveraging the distinct substrate specificity of A-domains, we previously developed photoaffinity probes for A-domains based on derivatization with a 5′-<em>O</em>-<em>N</em>-(aminoacyl)sulfamoyl adenosine (aminoacyl-AMS)-appended clickable benzophenone. Although our photoaffinity probes with different amino acid warheads enabled selective detection, visualization, and enrichment of target A-domains in proteomic environments, the effects of photoaffinity linkers have not been investigated. To explore the optimal benzophenone-based linker scaffold, we designed seven photoaffinity probes for the A-domains with different lengths, positions, and molecular shapes. Using probes <strong>2</strong>–<strong>8</strong> for the phenylalanine-activating A-domain of gramicidin S synthetase A (GrsA), we systematically investigated the binding affinity and labeling efficiency of the endogenous enzyme in a live producer cell. Our results indicated that the labeling efficiencies of probes <strong>2</strong>–<strong>8</strong> tended to depend on their binding affinities rather than on the linker length, flexibility, or position of the photoaffinity group. We also identified that probe <strong>2</strong> with a 4,4′-diaminobenzophenone linker exhibits the highest labeling efficiency for GrsA with fewer non-target labeling properties in live cells.</p></div>","PeriodicalId":255,"journal":{"name":"Bioorganic & Medicinal Chemistry","volume":null,"pages":null},"PeriodicalIF":3.3000,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Probing for optimal photoaffinity linkers of benzophenone-based photoaffinity probes for adenylating enzymes\",\"authors\":\"Sho Konno , Fumihiro Ishikawa , Hideaki Kakeya , Genzoh Tanabe\",\"doi\":\"10.1016/j.bmc.2024.117815\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>The adenylation (A) domain of non-ribosomal peptide synthetases (NRPSs) catalyzes the adenylation reaction with substrate amino acids and ATP. Leveraging the distinct substrate specificity of A-domains, we previously developed photoaffinity probes for A-domains based on derivatization with a 5′-<em>O</em>-<em>N</em>-(aminoacyl)sulfamoyl adenosine (aminoacyl-AMS)-appended clickable benzophenone. Although our photoaffinity probes with different amino acid warheads enabled selective detection, visualization, and enrichment of target A-domains in proteomic environments, the effects of photoaffinity linkers have not been investigated. To explore the optimal benzophenone-based linker scaffold, we designed seven photoaffinity probes for the A-domains with different lengths, positions, and molecular shapes. Using probes <strong>2</strong>–<strong>8</strong> for the phenylalanine-activating A-domain of gramicidin S synthetase A (GrsA), we systematically investigated the binding affinity and labeling efficiency of the endogenous enzyme in a live producer cell. Our results indicated that the labeling efficiencies of probes <strong>2</strong>–<strong>8</strong> tended to depend on their binding affinities rather than on the linker length, flexibility, or position of the photoaffinity group. We also identified that probe <strong>2</strong> with a 4,4′-diaminobenzophenone linker exhibits the highest labeling efficiency for GrsA with fewer non-target labeling properties in live cells.</p></div>\",\"PeriodicalId\":255,\"journal\":{\"name\":\"Bioorganic & Medicinal Chemistry\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.3000,\"publicationDate\":\"2024-06-25\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Bioorganic & Medicinal Chemistry\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0968089624002293\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Bioorganic & Medicinal Chemistry","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0968089624002293","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
非核糖体肽合成酶(NRPSs)的腺苷酸化(A)结构域催化底物氨基酸和 ATP 的腺苷酸化反应。利用 A 结构域独特的底物特异性,我们先前开发出了基于 5'-O-N- (氨基酰)氨基磺酰基腺苷(aminoacyl-AMS)衍生化的 A 结构域光亲和探针。虽然我们的光亲和探针带有不同的氨基酸弹头,能够在蛋白质组环境中选择性地检测、显像和富集目标 A-结构域,但光亲和连接物的影响尚未得到研究。为了探索基于二苯甲酮的最佳连接剂支架,我们设计了七种不同长度、位置和分子形状的光亲和探针。我们使用针对绣线菊素 S 合成酶 A(GrsA)苯丙氨酸激活 A 结构域的探针 2-8 系统地研究了活体生产细胞中内源酶的结合亲和力和标记效率。结果表明,探针 2-8 的标记效率往往取决于它们的结合亲和力,而不是链接长度、灵活性或光亲和基团的位置。我们还发现,带有 4,4'-二氨基二苯甲酮连接体的探针 2 对 GrsA 的标记效率最高,而且在活细胞中的非目标标记特性较少。
Probing for optimal photoaffinity linkers of benzophenone-based photoaffinity probes for adenylating enzymes
The adenylation (A) domain of non-ribosomal peptide synthetases (NRPSs) catalyzes the adenylation reaction with substrate amino acids and ATP. Leveraging the distinct substrate specificity of A-domains, we previously developed photoaffinity probes for A-domains based on derivatization with a 5′-O-N-(aminoacyl)sulfamoyl adenosine (aminoacyl-AMS)-appended clickable benzophenone. Although our photoaffinity probes with different amino acid warheads enabled selective detection, visualization, and enrichment of target A-domains in proteomic environments, the effects of photoaffinity linkers have not been investigated. To explore the optimal benzophenone-based linker scaffold, we designed seven photoaffinity probes for the A-domains with different lengths, positions, and molecular shapes. Using probes 2–8 for the phenylalanine-activating A-domain of gramicidin S synthetase A (GrsA), we systematically investigated the binding affinity and labeling efficiency of the endogenous enzyme in a live producer cell. Our results indicated that the labeling efficiencies of probes 2–8 tended to depend on their binding affinities rather than on the linker length, flexibility, or position of the photoaffinity group. We also identified that probe 2 with a 4,4′-diaminobenzophenone linker exhibits the highest labeling efficiency for GrsA with fewer non-target labeling properties in live cells.
期刊介绍:
Bioorganic & Medicinal Chemistry provides an international forum for the publication of full original research papers and critical reviews on molecular interactions in key biological targets such as receptors, channels, enzymes, nucleotides, lipids and saccharides.
The aim of the journal is to promote a better understanding at the molecular level of life processes, and living organisms, as well as the interaction of these with chemical agents. A special feature will be that colour illustrations will be reproduced at no charge to the author, provided that the Editor agrees that colour is essential to the information content of the illustration in question.