Formation, Tailoring and Incorporation of Piperazates in Kutzneride Biosynthesis

描述（由申请人提供）：本提案将研究kutzneride家族天然产物的生物合成，更具体地说是非蛋白质氨基酸哌酸酯。胡椒酸酯是胡椒酸酯的aza类似物，可以作为六元脯氨酸转化模拟物，并以各种形式存在于许多非核糖体肽抗生素中。通过生物合成定制的脯氨酸模拟物的可用性可能导致新型肽天然产物类似物的生产。具体的目标集中在生物合成，功能化，并纳入到库兹奈德支架哌酸酯。更好地了解这些过程将导致切除的哌酸酯延伸结构域的发展。此外，这些延伸结构域的使用可以提供具有改进的抗生素或抗肿瘤性质的类似物。研究将集中在酶促N-N键形成和氧化剪裁的哌酸酯实体和拟建的哌酸A结构域的特异性。确定A结构域特异性需要合成各种前体，并通过与表达蛋白的ATP-PPi交换试验分析它们的掺入。随后的氨基酸结合分析将确定最有可能被相应的A结构域激活的哌酸酯衍生物。这些结果将有助于研究哌酸盐功能化的时间。对所提出的PipCAT结构域进行克隆、过表达和纯化，并与已知的PheATE或2-羟基-3,3-二甲基丁酸AKRT结构域结合，将产生二酮哌嗪和二酮tomorpholine产品，其结构与一类广谱抗菌剂cyclo（Phe-Pro）和cyclo（Leu-Pro）非常相似。胡椒酸酯与脯氨酸和胡椒酸酯的构象相似，使其成为未来研究的理想药效团。目前，人们对天然产物中哌酸酯的生物合成和掺入知之甚少。了解辣椒酸酯生物合成、裁剪和结合的机制将有助于生产各种非核糖体肽天然产物。最终，这些发现将产生重新设计蛋白质以提供新的抗菌药物的新方法。

DESCRIPTION (provided by applicant): This proposal will investigate the biosynthesis of the kutzneride family of natural products, and more specifically the nonproteinogenic amino acid piperazate. Piperazate, the aza-analogue of pipecolate, can serve as a six-membered proline turn mimic and is present in numerous nonribosomal peptide antibiotics in various forms. The availability of tailored proline mimics through biosynthesis may lead to the production of novel peptide natural product analogues. The specific aims focus on the biosynthesis, functionalization, and incorporation of piperazates into the kutzneride scaffold. A better understanding of these processes should lead to the development of excised piperazate elongation domains. Furthermore, usage of these elongation domains may furnish analogues with improved antibiotic or antitumor properties. Research will focus on the enzymatic N-N bond formation and oxidative tailoring of the piperazate entity and the specificity of the proposed piperazate A domain. Determination of the A domain specificity requires synthesis of various precursors and analysis of their incorporation through an ATP-PPi exchange assay with the expressed protein. Subsequent analysis of amino acid incorporation will determine the most likely piperazate derivative activated by the corresponding A domain. These results would aid in the investigation of the timing of piperazate functionalization. Cloning, overexpression, and purification of the proposed PipCAT domain and combination with the known PheATE or 2-hydroxy-3,3-dimethyl butyric acid AKRT domains would yield diketopiperazine and diketomorpholine products whose structure would closely mimic cyclo(Phe-Pro) and cyclo(Leu-Pro), a class of broad-spectrum antimicrobial agents. The conformational similarities of piperazate to that of proline and pipecolate make it a desirable pharmacophore for future research. Currently, very little is known about the biosynthesis and incorporation of piperazates in natural products. Understanding the mechanisms behind the biosynthesis, tailoring, and incorporation of piperazate would allow for the production of various nonribosomal peptide natural products. Ultimately, these findings would produce new methods for re-engineering proteins to furnish new antimicrobial agents. PUBLIC HEALTH RELEVANCE: Bacterial resistance to antibiotics is a longstanding problem in the healthcare field. This proposal focuses on understanding how specific enzymes operate and developing new methods for the production of unique antibiotics through genetic engineering. These advances could potentially lead to new drugs to overcome bacterial resistance.