Biochemical and Mechanistic Studies of Pseudoglycosyltransferases

描述(申请人提供)：这项拟议的工作描述了一条研究轨迹，以调查一组新发现的糖基转移酶(GT)样酶的遗传学、生物化学和催化机制，这些酶在次生代谢物生物合成中催化非糖苷C-N偶联。我们最近的研究表明，一些假定的GT可以识别激活的假糖(单糖的类似物，其中的环氧被亚甲基取代)作为底物。这些假糖转移酶，或“假糖基转移酶”(PsGT)，可能是从祖先的GT进化而来的，以至于它们可以识别非糖作为供体底物。然而，目前还没有直接的方法来迅速将它们与专门的GT区分开来。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严重障碍。该项目的总体目标是深入了解PsGT的催化功能，建立PsGT特有的遗传密码，从而能够快速准确地识别这类酶，并将其用作生物医学研究、药物发现和生物技术的工具。这项提议有两个具体目标。首先，我们提出了保留PsGT家族20酶VldE的分子机制。此前，我们已经对参与抗真菌药物validamcin生物合成的PsGT20(VldE)的生化功能进行了表征，并获得了它的晶体结构。此外，我们还从天蓝色链霉菌中构建了VldE和海藻糖6-磷酸合成酶(OtsA)的嵌合体，并对它们的功能进行了研究。我们将在这些研究的基础上，利用蛋白质工程(例如，点突变和片段替换)、底物模拟和动力学同位素效应研究以及额外的X射线结晶学进行详细的机制研究。其次，我们将确定可能的倒位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PsGT家族5(PsGT5)酶进行生化评估。此外，我们将通过基因组测序草案从相关的天然产物生产商中鉴定出其他PsGT5，如二形态链霉菌(Trestatin生产者)、聚合链霉菌(Amlostatin生产者)和粘生链霉菌ATCC 31305(寡聚抑素生产者)。我们将利用这些信息在PsGT和它们相应的GT之间进行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射线结晶学和化学方面的尖端技术来获取、研究和开发PsGts。这项研究的成功完成将在广泛的科学领域产生重大影响，如 开发的技术可能会促进产生有用化学实体的新方法，如碳水化合物模拟物、修饰的糖偶联物和可能有助于治疗人类疾病的新型生物活性天然产物。PI和Co-I一直在PsGTs的分子机制方面密切合作，该团队拥有完成这一项目的独特能力。我们相信，拟议的研究是非常有价值的，将加强我们机构的研究环境，并使学生接触到研究，与该领域(R15)计划的既定目标保持一致。

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that some putative GTs can recognize activated pseudosugars (analogues of monosaccharides in which the ring oxygen has been replaced by a methylene group) as substrates.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.