Probing the Aglycon Promiscuity of an Engineered Glycosyltransferase

Abstract

A sweet library: Two variants (wild‐type (WT) and a triple mutant) of glycosyltransferase (GT) OleD have been shown to catalyze glycosylation of over 70 substrates, formation of O‐, S‐ and N‐glycosidic bonds, and iterative glycosylation (see scheme). Identified substrates include nucleophiles not previously known to act in GT reactions and span numerous natural product and therapeutic drug classes.

Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

“…The OleD Ser132 equivalent in the plant flavonoid GT VvGT1 (Thr141) forms a hydrogen bond with the C-6 hydroxyl group of UDP-2-deoxy-2-fluoroglucose (33,34). Lack of a hydrogen bond donor/acceptor at this position may explain the large increases in activity observed in previous studies (17) and in this investigation, particularly with C-6 modified sugars.…”

“…The alteration of position 132 from serine to phenylalanine was initially reported by Williams et al (14), and the same alteration was present in a number of other characterized OleD variants (15)(16)(17)(18)(19). The OleD Ser132 equivalent in the plant flavonoid GT VvGT1 (Thr141) forms a hydrogen bond with the C-6 hydroxyl group of UDP-2-deoxy-2-fluoroglucose (33,34).…”

“…Substitution of residue Ala242 with hydrophobic replacements (Val, Leu, and Ile) has been reported in several cases to be advantageous in the context of both forward and reverse GT-catalyzed reactions (14)(15)(16)(17)(18)(19). In the OleD crystal structure, Ala242 follows Ser241, which forms a hydrogen bond to the α-phosphate of the UDP acceptor in the context of a GT-catalyzed reverse reaction (this interaction was also observed in the VvGT1 structure) (14,27,33).…”

“…The P67T mutation was initially identified from a random mutagenesis library screened in the context of a GT-catalyzed forward reaction (14) and has been further studied in conjunction with additional OleD variants (15)(16)(17)(18)(19). Within the reported OleD crystal structure (27), residue 67 is situated amid a loop region (amino acids 60-76, loop N3), which is hypervariable in other GTs possessing the GT-B fold and contributes to forming the "donor" site in the context of GT-catalyzed reverse reactions (14,31).…”

“…However, many glycosyltransferase (GT)-catalyzed reactions are known to be readily reversible, enabling the "pirating" of unique sugars from natural products or alternative donors (resulting in generation of the respective sugar nucleotide) and one-pot sugar exchange reactions between unique natural products (4,(10)(11)(12)(13). This general reaction feature, in conjunction with availability of highly permissive glycosyltransferases (14)(15)(16)(17)(18) and simple donors designed to fundamentally alter the reaction thermodynamics, recently enabled a unique platform for NDP-sugar synthesis and a high throughput colorimetric screen for NDP-sugar formation and utilization (19). While the prior platform proof-of-concept study highlighted the syntheses of 22 natural and nonnatural TDP/UDP-sugars from 11 distinct 2-chloro-4-nitrophenyl glycoside donors using a single GT catalyst (Fig.…”