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that some putative GTs can recognize activated pseudosugars (analogues of monosaccharides in which the ring oxygen has been replaced by a methylene group) as substrates.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.
This proposal describes cutting-edge investigations of the genetics, biochemistry, and molecular mechanism of a newly recognized group of enzymes, called the pseudoglycosyltransferases (PsGTs), which are highly similar to the glycosyltransferases but catalyze nonglycosidic C-N couplings in the biosynthesis of bioactive natural products. Successful outcomes of this research will lead to the establishment of a new family of enzymes that may be utilized as tools for producing carbohydrate mimetics, redesigning glycoconjugates, creating new bioactive natural products, and discovering and developing new drugs. The proposed research is highly meritorious and the AREA (R15) grant will strengthen the research environment of the institution and expose undergraduate, professional, and graduate students to research.