STRUCTURE OF PEPTIDE SYNTHETASES AND RELATED ENZYMES

肽合成酶及相关酶的结构

• 批准号: 7250250
• 负责人: ANDREW M GULICK
• 金额: $ 29.54万
• 依托单位: HAUPTMAN-WOODWARD MEDICAL RESEARCH INST
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7250250
• 关键词: 4-chlorobenzoate-CoA ligase; Acetate-CoA Ligase; Adopted; Amino Acids; Anabolism; Antibiotics; Binding; Biochemical; Biological Factors; C-terminal; Carrier Proteins; Catalytic Domain; Chemicals; Class; Coenzyme A; Development; Employee Strikes; Engineering; Enzymes; Escherichia coli; Escherichia coli Proteins; Exhibits; Family; Foundations; Genetic; Genetic Engineering; Individual; Investigation; Length; Ligands; Ligase; Link; Modeling; Modification; Molecular Conformation; Mutation; Pantetheine; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Proteins; Reaction; Roentgen Rays; Rotation; Structure; Sulfhydryl Compounds; System; Tertiary Protein Structure; Work; adenylate; anticancer activity; catalyst; combinatorial; design; insight; interest; novel; novel strategies; peptide synthase; polypeptide; research study; thioester

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.

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目标，以创建可以产生新型肽的催化剂。 NRPS 是模块化蛋白质，包含表达为单个多肽的多个催化结构域。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，以进一步延伸或化学修饰。我们确定了两种腺苷酸形成酶的 X 射线晶体结构，表明在反应的不同步骤，C 末端结构域的方向相差 150 度。我们提出，密切相关的 NRPS 腺苷酸化结构域（激活氨基酸构建块并将其共价连接到第二个 NRPS 载体蛋白结构域）也采用这两种构象。这种变化的幅度和使用这些酶的方式是惊人的，并且表明改造 NRPS 酶以制造新型药物的努力将需要采取措施避免因下游结构域旋转而引起的空间冲突。 这种结构域交替假说将通过对三种腺苷酸形成酶（包括三结构域 NRPS）的 X 射线晶体学和生化分析进行研究。具体来说，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，我们已在活性结构域中表达了截短的两结构域腺苷酸化结构域-载体蛋白结构域片段。形式，和 d) 我们已经结晶的双结构域 NRPS 蛋白，它充当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，为设计这些催化剂以开发新药提供结构基础。