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.

Figure 1. Relevant GT-catalyzed coumarin glycosylation reactions

(A) The reaction catalyzed by WT NovM. (B) The reaction employed for the fluorescence-based screening assay used to evolve OleD. (C) Representation of the novobiocic acid glucosylation reaction catalyzed by WT and variant OleD. (D) The structures of representative naturally-occurring aminocoumarin antibiotics novobiocin (8), clorobiocin (9) and coumermycin A₁ (10).

Figure 2. Creation of OleD variants improved toward novobiocic acid (1)

(A) Specific activities of WT and mutant OleDs with novobiocic acid (1) and UPD-Glc (5) as acceptor/donor, respectively. (B) The crude cell extract glucosylation activities of randomly selected colonies from saturation mutagenesis libraries P67X, I112X, and A242X, with 1 as acceptor. Activities are illustrated in descending order and arrows designate clones that were selected for in-depth characterization.

Figure 3. Steady-state kinetic analysis of WT and P67T/I112K/A242V OleD

(A) WT OleD with novobiocic acid (1) as variable substrate and [UDP-Glc (5)] fixed at 5 mM. (B) WT OleD with UDP-Glc (5) as variable substrate and [novobiocic acid (1)] fixed at 5 mM. (C) P67T/I112K/A242V OleD with novobiocic acid (1) as variable substrate and [UDP-Glc (5)] fixed at 5 mM. (D) P67T/I112K/A242V OleD with UDP-Glc (5) as variable substrate and [novobiocic acid (1)] fixed at 5 mM.

Figure 4. Probing the donor specificity of WT OleD and mutant prodigy

(A) The set of UDP-sugar donors used to probe specificity. Dashed-boxed donors were detectable substrates for both WT and mutant P67T/I112T/A242V OleD, while solid boxed donors were substrates only for P67T/I112T/A242V. (B) Successful conversion rates (%) after 3 h with WT or P67T/I112T/A242V OleD using 50 μM acceptor 1 and 250 μM UDP-sugar donors (the reactions containing 28 were incubated for 18 h). (C) Improvement of donor promiscuity with increasing proficiency of OleD variants.

Figure 5. OleD active site structure

The key residues delineated in this study are highlighted within the previously reported active site structure of OleD bound to oleandomycin and NDP (PDB file 21YF). Color designations – substrates, orange; Pro-67, red; Ile-112, cyan; Ser-132, magenta; loop N3, yellow; dashed line, H-bond between the catalytic His-25 and acceptor sugar-OH. Residues in green are those that form the acceptor binding pocket, which is largely hydrophobic.