Characterization of Catalytic Diversity in Megaenzyme Systems

. 细菌和真菌产生广泛的次级代谢产物。这些天然化学物质使微生物具有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试剂、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产物的蛋白质构成称为聚酮酶和非核糖体肽合成酶的大型复杂酶系统（巨酶或巨合成酶）。典型的大合成酶由特定结构域的重复组合形成，并从单体结构单元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挑选蛋白质、蛋白质结构域和/或底物来进一步增加多样性。这项研究计划的目标是调查和结构表征megaenzyme系统，显示非典型的组成和机制，导致这些新的组件。 我们将研究的前两个不寻常的巨酶系统是寡核苷酸合酶和缩肽脱氢酶。Andrimid合酶是一种巨酶，它将转氨酶同系物AdmF纳入其合成循环。转氨酶通常与血液凝固和蛋白质交联相关，但AdmF是一种独立的蛋白质，其催化与巨酶载体蛋白结合的脂肪酸和氨基酸的连接。缩肽，如催泪素和抗霉素，是单体之间具有酰胺和酯键的小分子。缩肽脱氢酶使用α-酮酸作为底物并在增选的酮还原酶结构域中还原它们以产生α-羟基酸以掺入其产物中。 我们将使用X射线晶体学，突变和动力学分析，以及其他生物化学和生物物理学方法研究非经典巨酶系统的结构和功能。这将提供用于制造不同化学产品的机制的基本理解。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品。

. 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.