Biochemical and Mechanistic Studies of Pseudoglycosyltransferases

描述（由申请人提供）：拟议的工作描述了一个研究轨迹，以调查一组新认识的糖基转移酶（GT）样酶的遗传学、生物化学和催化机制，这些酶催化次级代谢物生物合成中的非糖苷C-N偶联。我们最近的研究表明，一些推定的GT可以识别活化的假糖（类似物的单糖，其中环氧已被取代的亚甲基）作为底物。这些假糖基转移酶，或“假糖基转移酶”（PsGT），可能已经从祖先GT进化到它们可以识别非糖作为供体底物的程度。然而，目前还没有直接的方法来快速区分它们与专用GT。这种知识的缺乏阻碍了它们的全部潜力的开发，并可能对糖科学，药物发现和相关领域的进展构成关键障碍。该项目的总体目标是深入了解PsGTs的催化功能，建立PsGTs独特的遗传密码，从而能够快速准确地识别这类酶，并利用它们作为生物医学研究，药物发现和生物技术的工具。这项建议有两个具体目标。首先，我们建议探索保留PsGT家族20酶VldE的分子机制。在此之前，我们已经表征了PsGT 20（VldE）的生化功能，它参与了抗真菌剂井冈霉素的生物合成，并获得了它的晶体结构。此外，我们已经创建了嵌合体的VldE和海藻糖6-磷酸合酶（OtsA）从天蓝色链霉菌和其功能的特点。我们将采用蛋白质工程（例如，点突变和片段置换），底物类似物和动力学同位素效应研究，以及额外的X射线晶体学。其次，我们将建立假定的倒位PsGTs的功能及其遗传特征。我们将对参与抗糖尿病药物阿卡波糖生物合成的推定PsGT家族5（PsGT 5）酶进行生物化学评价。此外，我们还将从相关的天然产物生产者中鉴定其他PsGT 5，如Streptomyces dimorphogenes（一种曲他汀生产者），S.产淀粉酶抑制素的S. lobatus;粘液生成菌ATCC 31305（一种寡抑素生产者）。我们将使用这些信息进行PsGT及其相应GT之间的比较生物信息学分析。该项目采用多学科方法，利用分子遗传学，蛋白质工程，X射线晶体学和化学的尖端技术来访问，研究和利用PsGT。这项研究的成功完成将在广泛的科学领域产生重大影响， 所开发的技术可促进产生有用的化学实体的新方法，所述化学实体例如可用于治疗人类疾病的碳水化合物模拟物、修饰的糖缀合物和新的生物活性天然产物。PI和Co-I一直在PsGT的分子机制方面密切合作，该团队具有完成该项目的独特能力。我们相信，拟议的研究是非常值得的，并将加强我们机构的研究环境，让学生接触研究，与区域（R15）计划的既定目标一致。

DESCRIPTION (provided by applicant): The proposed work describes a research trajectory to investigate the genetics, biochemistry, and catalytic mechanism of a newly recognized group of glycosyltransferase (GT)-like enzymes that catalyze nonglycosidic C-N couplings in secondary metabolite biosynthesis. Our recent study revealed that some putative GTs can recognize activated pseudosugars (analogues of monosaccharides in which the ring oxygen has been replaced by a methylene group) as substrates. These pseudosugar-transferase enzymes, or "pseudoglycosyltransferases" (PsGTs), might have evolved from ancestral GTs to the extent that they can recognize non-sugars as donor substrates. However, there is currently no straightforward way to rapidly discriminate them from dedicated GTs. This lack of knowledge hinders the exploitation of their full potential and may pose critical barriers to progress in glycoscience, drug discovery, and related fields. The overall goals of this project are to gain insights into the catalytic functions of PsGTs, to establish genetic codes unique for PsGTs that will enable quick and accurate identification of this class of enzymes, and to exploit their utilit as tools for biomedical research, drug discovery, and biotechnology. This proposal has two specific aims. First, we propose to explore the molecular mechanism of the retaining PsGT family 20 enzyme, VldE. Previously, we have characterized the biochemical function of a PsGT20 (VldE), which is involved in the biosynthesis of the antifungal agent validamycin, and obtained its crystal structure. In addition, we have created chimeras of VldE and a trehalose 6-phosphate synthase (OtsA) from Streptomyces coelicolor and characterized their functions. We will follow up these studies with detailed mechanistic investigations employing protein engineering (e.g., point mutations and fragment replacement), substrate analog and kinetic isotope effect studies, as well as additional X-ray crystallography. Second, we will establish the functions of putative inverting PsGTs and their genetic characteristics. We will carry out biochemical evaluation of the putative PsGT family 5 (PsGT5) enzyme involved in the biosynthesis of the antidiabetic drug acarbose. In addition, we will identify other PsGT5s from related natural product producers, such as Streptomyces dimorphogenes (a trestatin producer), S. conglobatus (an amylostatin producer), and S. myxogenes ATCC 31305 (an oligostatin producer), by draft genome sequencing. We will use the information for comparative bioinformatic analysis between PsGTs and their corresponding GTs. This project employs a multidisciplinary approach that utilizes cutting-edge technologies in molecular genetics, protein engineering, X-ray crystallography, and chemistry to access, study, and exploit PsGTs. The successful completion of this research will have significant impacts in broad scientific fields, as the technology developed may facilitate new ways of generating useful chemical entities, such as carbohydrate mimetics, modified glycoconjugates and novel bioactive natural products that may be useful in the treatment of human diseases. The PI and Co-I have been working closely together on the molecular mechanism of PsGTs and the team has the unique capability to complete this project. We believe that the proposed research is highly meritorious and will strengthen the research environments of our institutions and expose students to research, consistent with the stated goals of the AREA (R15) program.