Characterization of Catalytic Diversity in Megaenzyme Systems

大酶系统催化多样性的表征

• 批准号: 418420-2012
• 负责人: Schmeing, ThomasMartin
• 金额: $ 2.26万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=630045
• 关键词: Characterization; Catalytic; Diversity; Megaenzyme; Systems

. Bacteria and fungi produce a wide range of secondary metabolites. These natural chemicals give microbes growth advantages or kill competing organisms. The biological activities of these compounds also make them useful to society as agricultural agents, pesticides and "green" chemicals. Many of the proteins responsible for synthesis of these secondary metabolites constitute large, complex enzyme systems (megaenzymes or megasynthases) called polyketide synthases and nonribosomal peptide synthetases. Canonical megasynthases are formed of repeating combinations of specific domains, and produce a variety of small molecule compounds from monomer building blocks. Intriguingly, in some systems, the diversity is further increased by co-opting proteins, protein domains, and/or substrates from various other cellular processes. The goal of this research program is to investigate and structurally characterize megaenzyme systems that display atypical composition and mechanisms resulting from the inclusion of these novel components. The first two unusual megaenzyme systems we will investigate are andrimid synthase and the depsipeptide synthases. Andrimid synthase is a megaenzyme that has co-opted a transglutaminase homologue, AdmF, into its synthetic cycle. Transglutaminases are usually associated with blood clotting and protein crosslinking, but AdmF is a free-standing protein that catalyzes the joining of a fatty acid and an amino acid which are bound to megaenzyme carrier proteins. Depsipeptides, such as cereulide and antimycin, are small molecules with amide and ester linkages between monomers. Depsipeptide synthases use a-keto acids as substrates and reduce them in a co-opted ketoreductase domain to produce a-hydroxy acids to incorporate into their product. We will study the structure and function of noncanonical megaenzyme systems using X-ray crystallography, mutational and kinetic analysis, and other biochemical and biophysical approaches. This will provide fundamental understanding of the mechanisms employed to make diverse chemical products. These mechanisms could possibly be exploited to produce novel biochemicals for use in industry and agriculture.

。细菌和真菌产生广泛的次生代谢产物。这些天然化学物质给微生物带来生长优势，或者杀死竞争对手的有机体。这些化合物的生物活性也使它们成为对社会有用的农用制剂、杀虫剂和“绿色”化学品。许多负责合成这些次级代谢物的蛋白质组成了大型、复杂的酶系统(巨酶或巨合酶)，称为聚酮合成酶和非核糖体多肽合成酶。典型的巨合酶是由特定结构域的重复组合形成的，并从单体构建块产生各种小分子化合物。有趣的是，在一些系统中，通过加入来自各种其他细胞过程的蛋白质、蛋白结构域和/或底物，进一步增加了多样性。这项研究计划的目标是调查和结构表征巨型酶系统，这些系统表现出非典型的组成和机制，由这些新的成分所导致。我们要研究的前两个不同寻常的巨酶系统是雄激素合成酶和去脂肽合成酶。安德里米合成酶是一种巨型酶，它将转谷氨酰胺酶同系物AdmF纳入其合成周期。转谷氨酰胺酶通常与血液凝固和蛋白质交联有关，但AdmF是一种独立的蛋白质，它催化与巨酶载体蛋白结合的脂肪酸和氨基酸的连接。谷氨酰胺和抗菌素是单体间具有酰胺和酯键的小分子。深度肽合成酶使用a-酮酸作为底物，并在增选的酮基还原酶结构域中将其还原，以产生a-羟基酸，并将其并入其产品中。我们将使用X射线结晶学、突变和动力学分析以及其他生化和生物物理方法来研究非典型巨酶系统的结构和功能。这将提供对用于制造不同化学产品的机理的基本理解。这些机制可能被利用来生产用于工业和农业的新型生物化学品。