Distinct Substrate Specificity and Catalytic Activity of the Pseudoglycosyltransferase VldE

Abstract

SUMMARY The pseudoglycosyltransferase (PsGT) VldE is a glycosyltransferase-like protein that is similar to trehalose 6-phosphate synthase (OtsA). However, in contrast to OtsA, which catalyzes condensation between UDP-glucose and glucose 6-phosphate, VldE couples two pseudosugars to give a product with an α,α-N-pseudoglycosidic linkage. Despite their unique catalytic activity and important role in natural products biosynthesis, little is known about the molecular basis governing their substrate specificity and catalysis. Here, we report comparative biochemical and kinetic studies using recombinant OtsA, VldE, and their chimeric proteins with a variety of sugar and pseudosugar substrates. We found that the chimeric enzymes can produce hybrid pseudo-(amino)disaccharides and an amino group in the acceptor is necessary to facilitate a coupling reaction with a pseudosugar donor. Furthermore, we found that the N-terminal domains of the enzymes not only play a major role in selecting the acceptors, but also control the type of nucleotidyl diphosphate moiety of the donors.

Biosynthesis of nitrogen-containing natural products, C7N aminocyclitols and bis-indoles, from actinomycetes

“…Interestingly, VldE did not accept GDP-glucose and glucose 6-phosphate as substrates to produce trehalose 6-phosphate, which indicated the narrow substrate tolerance of VldE, a dedicated enzyme for validamycin A biosynthesis. 44,45) Upon the addition of VldH, consumption of validoxylamine A 7′-phosphate and formation of validoxylamine A was observed, which clearly demonstrated the late enzymatic conversion steps in validamycin A biosynthesis. 44) To investigate how the VldE enzyme transfers a nonsugar donor molecule (GDP-activated C 7 cyclitol) to the acceptor molecule (a non-sugar molecule with an amine group) with the net retention of stereochemistry, a series of VldE crystal structures were solved and analyzed.…”

“…Analogous to the proposed reaction mechanism for retaining-type glycosyltransferases, 42) hydrogen bonding interaction from the donor phosphate group to the acceptor nucleophile was proposed to enable front side attack to promote a substitution reaction while retaining its configuration, and the double bond π-electron of the donor non-sugar was predicted to mimic the transition state in the PsGT reaction. [44][45][46] Further studies showed the importance of the amine group as a better nucleophile to promote the coupling reaction 45) (Fig. 3).…”

“…Based on the crystal structures, Abuelizz and Mahmud produced the domain-swapped chimeric proteins of VldE and OtsA from S. coelicolor A3(2) successfully, and examined the activity of these "chimeras" with combinations of substrates, to elucidate their substrate tolerances. 45) By swapping the substrates, they showed the potential as biocatalysis of engineered proteins, and the study also showed the importance of the amine group as a better nucleophile to promote the coupling reaction. 45) The VldE reaction in validamycin A biosynthesis is the first and the only characterized reaction for a PsGT.…”

“…45) By swapping the substrates, they showed the potential as biocatalysis of engineered proteins, and the study also showed the importance of the amine group as a better nucleophile to promote the coupling reaction. 45) The VldE reaction in validamycin A biosynthesis is the first and the only characterized reaction for a PsGT. Analogous to the proposed reaction mechanism for retaining-type glycosyltransferases, 42) hydrogen bonding interaction from the donor phosphate group to the acceptor nucleophile was proposed to enable front side attack to promote a substitution reaction while retaining its configuration, and the double bond π-electron of the donor non-sugar was predicted to mimic the transition state in the PsGT reaction.…”

Biosynthesis and metabolic engineering of pseudo-oligosaccharides

“…One of the most intriguing questions related to the biosynthesis of pseudo-oligosaccharides asks how the pseudosugar is attached to the acceptor to form a C-N bond in those compounds. Detailed bioinformatic and biochemical studies on the validamycin pathway revealed that a glycosyltransferase-like protein (VldE/ValL) is responsible for the formation of the nonglycosidic C-N bond in validoxylamine A using GDP-valienol as a donor pseudosugar and validamine 7-phosphate as an acceptor pseudosugar (Figure 5) (44, 45). The product, validoxylamine A 7´-phosphate, is then converted to validoxylamine A by the phosphatase VldH.…”