Optimizing Glycosyltransferase Specificity via “Hot Spot” Saturation Mutagenesis Presents a Catalyst for Novobiocin Glycorandomization

Abstract

A comprehensive two-phase "hot spot" saturation mutagenesis strategy for the rapid evolution of glycosyltransferase (GT) specificity for nonnatural acceptors is described. Specifically, the application of a high-throughput screen (based on the fluorescent acceptor umbelliferone) was used to identify key amino acid hot spots that contribute to GT proficiency and/or promiscuity. Saturation mutagenesis of the corresponding hot spots facilitated the utilization of a lower-throughput screen to provide OleD prodigy capable of efficiently glycosylating the nonnatural acceptor novobiocic acid with an array of unique sugars. Incredibly, even in the absence of a high-throughput screen for novobiocic acid glycosylation, this approach rapidly led to improvements in the desired catalytic activity of several hundred-fold.

Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

“…While mutations at position 112 have been previously characterized (14,15,18), advantageous mutations of position 113 within OleD have not been reported. The single mutation I112T was initially reported as having no effect upon the OleD-catalyzed glucosylation of 4-methylumbelliferone (14), but a subsequent study noted substantial improvement of k cat /K m upon the OleDcatalyzed glucosylation of novobiocic acid by both I112T and I112K (15).…”

“…The single mutation I112T was initially reported as having no effect upon the OleD-catalyzed glucosylation of 4-methylumbelliferone (14), but a subsequent study noted substantial improvement of k cat /K m upon the OleDcatalyzed glucosylation of novobiocic acid by both I112T and I112K (15). In the context of a GT-catalyzed reverse reaction, residues 112 and 113 are located deep within the donor pocket ( Fig.…”

“…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.…”

“…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.…”