Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

Abstract

We described the integration of the general reversibility of glycosyltransferase-catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial sugar nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed maximum improvements in k cat /K m of >400-fold and >15-fold for formation of NDP-glucoses and UDP-sugars, respectively. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2-chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP-sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex sugar nucleotides and the practical synthesis of a unique set of sugar nucleotides.carbohydrate | enzyme | glycobiology | protein engineering T he lack of accessibility and availability of uncommon and uniquely functionalized sugar nucleotides (NDP-sugars) continues to restrict research focused upon understanding the regulation, biosynthesis, and/or role of glycosylated macromolecules and glycosylated small molecules in biology or therapeutic development (1-7). Although there are many reported chemical, enzymatic, and chemoenzymatic strategies for NDP-sugar synthesis, those that extend beyond the reach of common biological sugars (e.g., Dglucose, D-galactose, etc.) nearly all suffer from long reaction times (>16 h), relatively low yields, and difficulties associated with product purification and/or stability (3,4,8,9). Thus, the development of robust methods for sugar nucleotide synthesis directly compatible to the downstream biological processes to be studied may be advantageous.From a traditional viewpoint, NDP-sugars are used as donors by Leloir glycosyltransferases (sugar nucleotide-dependent enzymes) for formation of glycosidic bonds. 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, 10-13). This general reaction feature, in conjunction with availability of highly permissive glycosyltransferases (14-18) 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. 1A) (19), the substrate specificity of the glycosyltransferase used ...

Enzymatic Biosynthesis of Novel Resveratrol Glucoside and Glycoside Derivatives

“…Among these, some have shown enhanced biological properties compared to their respective parent molecules (44). In addition, glycosyltransferases (GTs) (e.g., oleandomycin GT [OleD] and its variants [49,50], kanamycin GT-KanE, and vancomycin GT-GtfE [51]) have been engineered to increase their flexibility in accepting various donor and acceptor substrates. For the present study, a GT, YjiC (GenBank accession no.…”

A Simple Strategy for Glycosyltransferase‐Catalyzed Aminosugar Nucleotide Synthesis

“…An overall preference for glucosides (rank order of 6-NH 2 ≈ 4-NH 2 ≈ 2-NH 2 > 3-NH 2 ) over xylosides (rank order of 4-NH 2 ≈ 2-NH 2 ) was observed with no apparent difference between the donor free base and the corresponding hydrochloride salt (Table S2, Figure S2, S3, S4, S5). By comparison, both wtOleD and OleD TDP-16 were notably worse than OleD Loki with one exception (6-deoxy-6-azido-β-D-glucoside 2 ), a previously reported substrate of TDP-16, [15a] where TDP-16 was found to slightly outperform OleD Loki in this endpoint assay. In addition, OleD Loki displayed notable improvement with additional non-native donors beyond the scope of the targeted aminosugar series including 6-deoxy-6- N -acetylamino-β-D-glucoside 19 and slight improvement with α-L-arabinoside 18 - both analogs generated during the course of synthetic methods development.…”

“…[15] Herein we describe an interrogation of two of the most permissive glycosyltransferase prodigy from these prior studies (OleD TDP-16 – a P67T/S132F/A242L/Q268V variant; [15a,16] OleD Loki – a P67T/I112P/T113M/S132F/A242I variant [15b] ) for their abilities to catalyze the production of variant aminopentose and/or aminohexose nucleotides in the presence of the corresponding 2-chloro-4-nitrophenyl aminosugar donors and TDP/UDP. This study reveals OleD Loki to catalyze the conversion of 6 out of 7 simple D-glucosamino- and D-xylosamino-glycoside donors into their corresponding UDP/TDP-aminosugar nucleotides and also to utilize 6-azido/acetylamino-D-glucoside donors.…”

“…Of these, TDP-16 [16] and Loki [15b] are engineered variants of the Streptomyces antibioticus macrolide glucosyltransferase (wtOleD). [19] Standard conditions (10 µM OleD variant, 2 mM UDP or 5 mM TDP, 2 mM 2-chloro-4-nitophenyl glycoside, 50 mM Tris-HCl, pH 8.0, final volume of 100 µL, 25µC, 12 h followed by the RP HPLC analysis) were utilized to compare the turnover across the entire panel of enzyme/glycoside combinations.…”

“…[22] The current strategy affords the desired UDP/TDP-aminosugars in 7%–28% yield (including the simple four-step synthesis from peracylated azidosugars). Furthermore, given OleD Loki was evolved to also efficiently utilize ADP, CDP, and GDP, [15b] the current study suggests the potential to also employ OleD Loki for the corresponding syntheses of ADP-, CDP-, and/or GDP-aminosugars.…”