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（NRPSS）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，以创建可以产生新肽的催化剂。 NRPS是模块化蛋白，包含以单个多肽表示的多个催化结构域。在合成过程中，新生肽从一个催化结构域转移到另一个催化域，以进行进一步的伸长或化学修饰。我们已经确定了两种腺苷酸形成酶的X射线晶体结构，这些酶表明，在反应的不同步骤中，C末端结构域的方向差异150度。我们提出，密切相关的NRP腺苷酸化结构域，该结构域激活氨基酸的构建块并共价连接到第二个NRPS载体蛋白结构域中，也采用了这两个构型。这些酶使用的大小和方式，这种变化令人震惊，并表明为制造NRPS酶制造新型药物的努力将要求采取步骤以避免由下游域旋转引起的空间冲突。 该域交替假设将通过三种腺苷酸形成酶（包括三域NRP）的X射线晶体学和生化分析进行研究。 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 领域。通过我们的结构性工作和生化分析，我们将深入了解这些重要的NRP领域的催化机制，为努力为这些催化剂设计新药提供了努力的结构基础。