Record Details
Mechanistic Studies and Catalytic Engineering of the Pseudoglycosyltransferase VldE
ScholarsArchive at Oregon State University
| Field | Value |
|---|---|
| Title | Mechanistic Studies and Catalytic Engineering of the Pseudoglycosyltransferase VldE |
| Names |
Abuelizz, Hatem A.
(creator) Mahmud, Taifo (advisor) |
| Date Issued | 2015-05-26 (iso8601) |
| Note | Graduation date: 2015 |
| Abstract | This thesis describes an investigation of the molecular basis of the substrate specificity and catalytic mechanism of a unique pseudoglycosyltransferase (PsGT) enzyme, VldE. PsGTs are a newly discovered group of glycosyltransferase (GT)-like proteins that catalyze the transfer of a pseudosugar to an acceptor molecule via a C-N bond formation. This inspiring pseudoglycosidic C-N coupling is involved in the biosynthesis of pseudosugar-containing natural products, such as the α-glycosidase inhibitors acarbose and amylostatins, the antibiotic pyralomicin 1a, and the antifungal agent validamycin A. The validamycin PsGT VldE is a GT-like protein similar to trehalose 6-phosphate synthase (OtsA). OtsA catalyzes a coupling reaction between UDP-glucose and glucose 6-phosphate to yield α,α-1,1-trehalose 6-phosphate. In contrast to OtsA, VldE catalyzes a coupling reaction between two pseudosugar substrates, GDP-valienol and validamine 7-phosphate, to give validoxylamine A 7'-phosphate with an α,α-N-pseudoglycosidic linkage. In spite of its unique catalytic function, little is known about the molecular basis governing its substrate specificity and reaction mechanism. To understand the molecular basis for the substrate specificity and catalytic activity of VldE we first examined the role of the N- and C-terminal domains of the protein in its substrate recognition and catalysis. This was pursued by comparative biochemical and kinetic studies using VldE, OtsA and two chimeric proteins, which were produced by swapping the N- and C-terminal domains of VldE with those of OtsA from Streptomyces coelicolor. The wild-type enzymes and the resulting chimeric proteins were tested using a variety of synthetically prepared sugar (pseudosugar) donor and acceptor substrates. The results showed that both substrate specificity and acceptor molecule nucleophilicity play a role in the catalytic activity of VldE. Moreover, we found that the N-terminal domains of OtsA and VldE not only play a major role in selecting the sugar (pseudosugar) acceptors, but also control the type of nucleotidyl diphosphate moiety of the sugar (pseudosugar) donors. The study provided new insights into the distinct substrate specificity and catalytic activities of OtsA and VldE, and demonstrated that a C-1 amino group of the acceptor is necessary for the PsGT coupling reaction to occur. This result explains why all known natural products whose formations involve a PsGT contain amino linkage. The second part of the study addressed a fundamental question as to how VldE catalyzes a coupling between two non-sugar molecules with net retention of the ‘anomeric’ configuration of the donor cyclitol in the product. The current paradigm suggests that retaining GTs, such as GT family-5 (glycogen synthase) and GT family-20 (OtsA), function via an internal return (S[subscript N]i)-like mechanism involving an oxocarbenium ion-like transition state. However, as the donor substrate for VldE is an activated pseudosugar (GDP-valienol), oxocarbenium ion formation is not possible in the VldE-catalyzed coupling reaction. Taking advantage of information from the X-ray crystal structures of VldE, additional chimeric and point mutant proteins, were produced and tested. The study revealed a significant role of the N-terminal domains of OtsA and VldE in governing their substrate specificities, particularly for the acceptor molecules. In the case of VldE, the acceptor molecules need to have a strong nucleophilic group (amino group) at C-1, whereas OtsA needs an acceptor substrates with a hydroxyl group at C-1. Our work suggests that the distinct substrate specificities of OtsA and VldE are due to the differences in topology of the proteins particularly of the second half of the N-terminal domains (amino acids 128-266). Moreover, the study revealed a possible new reaction mechanism unique to the PsGT VldE and is different from the proposed (SNi)-like mechanism for the retaining GT OtsA. The new mechanism involves a deprotonation at C-7 of the unsaturated pseudosugar donor (GDP-valienol), followed by an electron shift of the ring olefin leading to 1,4-elimination of GDP. The oxocarbenium ion-like transition state geometry of GDP-valienol and the resemblance of the protein-ligand interactions with those of OtsA suggest that the C-N bond formation should occur via a front-face attack of the nucleophilic amino group of the acceptor molecule to give a product with net retention of the anomeric configuration. Combined together, the results not only provide insights into the molecular basis for the distinct substrate specificity and catalytic activity of VldE, but also present evidence for a potentially new catalytic mechanism unique to PsGT enzymes. |
| Genre | Thesis/Dissertation |
| Topic | Glycosyltransferases |
| Identifier | http://hdl.handle.net/1957/56101 |
