Characterization of Catalytic Diversity in Megaenzyme Systems

。细菌和真菌产生广泛的次生代谢物。这些天然化学物质赋予微生物生长优势或杀死竞争生物。这些化合物的生物活性也使它们作为农业药剂、杀虫剂和“绿色”化学品对社会有用。许多负责合成这些次级代谢物的蛋白质构成了大而复杂的酶系统（大型酶或大型合成酶），称为聚酮合成酶和非核糖体肽合成酶。典型的巨合酶是由特定结构域的重复组合形成的，并从单体构建块中产生各种小分子化合物。有趣的是，在一些系统中，通过从各种其他细胞过程中选择蛋白质、蛋白质结构域和/或底物，多样性进一步增加。本研究计划的目标是调查和结构表征巨型酶系统，显示非典型的组成和机制，导致这些新成分的包含。**我们将研究的前两个不寻常的巨型酶系统是雌雄同体合成酶和沉积肽合成酶。雌雄同体合成酶是一种大型酶，它选择了谷氨酰胺转氨酶同源物AdmF进入其合成周期。转谷氨酰胺酶通常与凝血和蛋白质交联有关，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结合。抑郁肽，如cereulide和抗霉素，是单体之间有酰胺和酯键的小分子。沉积肽合成酶使用a-酮酸作为底物，并在协同选择的酮还原酶域中还原它们，以产生a-羟基酸，并将其纳入其产物中。**我们将使用x射线晶体学、突变和动力学分析以及其他生化和生物物理方法研究非典型巨型酶系统的结构和功能。这将提供基本的机制，以使不同的化学产品的理解。这些机制可能被用来生产用于工业和农业的新型生物化学物质。＊＊＊＊

. 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. ****