Expanding the promiscuity of a natural-product glycosyltransferase by directed evolution

Abstract

Natural products, many of which are decorated with essential sugar residues, continue to serve as a key platform for drug development. Adding or changing sugars attached to such natural products can improve the parent compound's pharmacological properties, specificity at multiple levels, and/or even the molecular mechanism of action. Though some natural-product glycosyltransferases (GTs) are sufficiently promiscuous for use in altering these glycosylation patterns, the stringent specificity of others remains a limiting factor in natural-product diversification and highlights a need for general GT engineering and evolution platforms. Herein we report the use of a simple high-throughput screen based on a fluorescent surrogate acceptor substrate to expand the promiscuity of a natural-product GT via directed evolution. Cumulatively, this study presents variant GTs for the glycorandomization of a range of therapeutically important acceptors, including aminocoumarins, flavonoids and macrolides, and a potential template for engineering other natural-product GTs.

Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

“…Based upon this assessment, seven putative sugar donor-interacting positions (amino acids 67, 74, 85, 111-113, and 134) and eight potential NDP acceptor-interacting positions (amino acids 132, 242, 243, 268, 290, 309, 330, and 331) were selected for saturation mutagenesis to present the potential of 285 possible mutants to be generated for screening with each desired substrate pairing. Of these positions, five (amino acids 67, 112, 132, 242, and 268) were previously identified as impacting upon OleD permissivity and/or proficiency (9)(10)(11)(12)(13)(14). Special care was taken to ensure that the substrate binding pockets for both the putative 2-chloro-4-nitrophenyl glycoside donors (donors 1 and 4-8) and NDP acceptors were equally represented ( Fig.…”

“…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).…”

“…Thus, a representative OleD saturation library was generated. Positions targeted for saturation mutagenesis were selected by considering both previously identified mutational "hot spots" (14)(15)(16)18) and additional potential ligand-interacting residues based upon a model constructed from the structure of wild-type OleD (wtOleD) bound to erythromycin and UDP (27), wherein UDP-2-deoxy-2-fluoroglucose from the UGT72B1 (a GT possessing a similar GT-B fold to that of OleD; Protein Data Bank ID code 2VCE) ligand-bound structure (28) was substituted for UDP. Based upon this assessment, seven putative sugar donor-interacting positions (amino acids 67, 74, 85, 111-113, and 134) and eight potential NDP acceptor-interacting positions (amino acids 132, 242, 243, 268, 290, 309, 330, and 331) were selected for saturation mutagenesis to present the potential of 285 possible mutants to be generated for screening with each desired substrate pairing.…”

“…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.…”

Chemoenzymatic elaboration of monosaccharides using engineered cytochrome P450 BM3 demethylases

“…Elimination of protecting groups altogether also has been demonstrated using enzymes to affect the glycosylation of various substrates (8). The utility of this approach could be expanded greatly by identifying or engineering additional enzymes (9), but the preparation of substituted monosaccharides lies beyond its scope. Chemoenzymatic regioselective deprotection of a globally protected monosaccharide is a potentially practical alternative to these approaches.…”