Formation, Tailoring and Incorporation of Piperazates in Kutzneride Biosynthesis

描述（由申请人提供）：本提案将研究天然产物的kutzneride家族的生物合成，更具体地说是非蛋白质氨基酸哌嗪。哌可酯的氮杂类似物阿扎酯可作为六元脯氨酸转角模拟物，并以各种形式存在于许多非核糖体肽抗生素中。通过生物合成定制脯氨酸模拟物的可用性可能导致新的肽天然产物类似物的产生。具体的目标集中在哌嗪类化合物的生物合成、功能化和掺入到库茨内酯支架中。更好地了解这些过程应导致切除哌嗪延伸域的发展。此外，这些延伸结构域的使用可以提供具有改善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嗪实体和特异性的建议哌嗪A域。A结构域特异性的测定需要合成各种前体并通过与表达的蛋白质的ATP-PPi交换测定分析它们的掺入。随后的氨基酸掺入分析将确定最可能被相应的A结构域激活的哌嗪衍生物。这些结果将有助于研究哌嗪官能化的时机。所提出的PipCAT结构域的克隆、过表达和纯化以及与已知的PheATE或2-羟基-3，3-二甲基丁酸AKRT结构域的组合将产生二酮哌嗪和二酮吗啉产物，其结构将密切模拟环（Phe-Pro）和环（Leu-Pro），一类广谱抗微生物剂。 哌嗪酸酯与脯氨酸和哌啶酸酯的构象相似性使其成为未来研究的理想药效团。目前，对哌嗪类化合物的生物合成和在天然产物中的掺入知之甚少。了解背后的生物合成，剪裁和纳入哌嗪的机制将允许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的新方法，以提供新的抗菌剂。 公共卫生相关性：细菌对抗生素的耐药性是医疗保健领域的一个长期问题。该提案的重点是了解特定酶如何运作，并通过基因工程开发生产独特抗生素的新方法。这些进展可能会导致新的药物来克服细菌耐药性。

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.