Engineering novel S-glycosidase activity into extremo-adapted β-glucosidase by rational design

Abstract

The breakdown of sulphur glycosidic bonds in thioglycosides can produce isothiocyanate, a chemoprotective agent linked to the prevention of cancers, however only a handful of enzymes have been identified that are known to catalyse this reaction. Structural studies of the myrosinase enzyme, which is capable of hydrolysing the thioglycosidic bond, has identified residues that may play important roles in sulphur bond specific activity. Using rational design, two extremo-adapted β-glycosidases from the species Thermus nonproteolyticus (TnoGH1) and Halothermothrix orenii (HorGH1) were engineered towards thioglycoside substrates. Twelve variants, six for TnoGH1and six for HorGH1, were assayed for activity. Remarkable enhancement of the specificity (kcat/KM) of TnoGH1 and HorGH1 towards β-thioglycoside was observed in the single mutants TnoGH1-V287R (2500 M -1 s -1 ) and HorGH1-M229R, (13260 M -1 s -1 ) which showed a 3-fold increase with no loss in turnover rate when compared to the wild type enzymes . Thus, the role of arginine is key to induce β-thioglycosidase activity.Thorough kinetic investigation of the different mutants has shed light on the mechanism of β-glycosidases when acting on the native substrate. KEY POINTSKey residues were identified in the active site of Brevicoryne brassicae myrosinase.Rationally designed mutations were introduced into two extremo-adapted β-glycosidases. β-glycosidases mutants exhibited improved activity against thioglycosidic bonds.The mutation to arginine in the active site yielded the best variant.

Novel triple mutant of an extremophilic glycosyl hydrolase enables the rapid synthesis of thioglycosides

“…The high nucleophilicity of the thiolate tested with the double mutant scaffold stabilizing the transition states, 26,27 is indeed sufficient to achieve a significant product yield, but the active site is clearly positively impacted by the M299R mutation, as it was reported for the hydrolysis of thioglycosides. 36 Whereas the K M towards α- d -glucopyranosyl fluoride was found to be similar for the M299R/E166A/E354G and the E166A/E354G variants (2.26 and 2.12 mM, respectively), the K M towards p NT was lower for M299R/E166A/E354G (3.82 mM) than for E166A/E354G (7.46 mM) (Fig. S7, ESI†).…”

“…The HorGH1 M299R mutant exhibited a 3-fold increase in specificity for the hydrolysis of b-thioglycosides without any loss in turnover rate compared to the wild type (WT) enzymes. 36 The depletion of catalytic residues 26,27 combined with the HorGH1 M299R mutant (Fig. S1, ESI †) could yield a very powerful catalyst for the synthesis of still elusive thioglycosides.…”

“…The Hor GH1 M299R mutant exhibited a 3-fold increase in specificity for the hydrolysis of β-thioglycosides without any loss in turnover rate compared to the wild type (WT) enzymes. 36 …”