STRUCTURE OF PEPTIDE SYNTHETASES AND RELATED ENZYMES

描述（由申请人提供）：非核糖体肽合成酶（NRPS）产生具有抗生素和抗癌活性的肽，因此是组合或基因工程的靶标，以产生可产生新型肽的催化剂。NRPS是包含多个催化结构域的模块蛋白，其表达为单个多肽。在合成过程中，新生肽从一个催化结构域转移到下一个催化结构域以进行进一步延伸或化学修饰。我们已经确定了两种腺苷酸形成酶的X射线晶体结构，这表明在反应的不同步骤中，C-末端结构域的取向相差150度。我们已经提出，密切相关的NRPS腺苷酸化结构域，激活氨基酸的积木和共价连接到第二NRPS载体蛋白结构域，也采用这两种构象。这种变化的幅度和这些酶使用的方式是惊人的，并表明工程改造NRPS酶以制备新型药物的努力将需要采取步骤以避免由下游结构域的旋转引起的空间冲突。 这一结构域交替假说将通过X射线晶体学和生物化学分析的三个腺苷酸形成酶，包括一个三域NRPS。具体而言，我们将确定结构a）乙酰辅酶A合成酶，B）芳基辅酶A合成酶，c）三结构域NRPS蛋白，其中我们已经表达了活性形式的截短的两结构域腺苷酸化结构域-载体蛋白结构域片段，以及d）我们已经结晶的两结构域NRPS蛋白，其用作腺苷酸化结构域的氨基酸受体。通过我们的结构工作和生化分析，我们将深入了解这些重要的NRPS结构域的催化机制，为开发新药的催化剂提供结构基础。

DESCRIPTION (provided by applicant): Non-ribosomal peptide synthetases (NRPSs) produce peptides with antibiotic and anticancer activities and are therefore a target for combinatorial or genetic engineering to create catalysts that could generate novel peptides. NRPSs are modular proteins that contain multiple catalytic domains expressed as a single polypeptide. During synthesis, the nascent peptide is transferred from one catalytic domain to the next for further elongation or chemical modification. We have determined the X-ray crystal structures of two adenylate-forming enzymes that suggest that, at different steps of the reaction, the orientation of the C-terminal domain differs by 150 degrees. We have proposed that the closely-related NRPS adenylation domains, which activate the amino acid building blocks and covalently attach them to a second NRPS carrier protein domain, also adopt these two conformations. The magnitude of, and the manner in which these enzymes use, this change is striking and suggests that efforts to engineer the NRPS enzymes to make novel pharmaceuticals will require that steps are taken to avoid steric clashes that arise from the rotation of downstream domains. This domain alternation hypothesis will be investigated through x-ray crystallographic and biochemical analyses of three adenylate-forming enzymes, including a three-domain NRPS. Specifically, we will determine the structures a) acetyl-CoA synthetase, b) an aryl-CoA synthetase, c) a three-domain NRPS protein of which we have expressed a truncated two-domain adenylation domain-carrier protein domain fragment in an active form, and d) a two-domain NRPS protein, which we have crystallized, that serves as the amino acid acceptor for the adenylation domain. Through our structural work and biochemical analyses we will gain insight into the catalytic mechanism of these important NRPS domains, providing the structural foundation for efforts to engineer these catalysts for the development of new drugs.