Catechol glucosides act as donor/acceptor substrates of glucansucrase enzymes of Lactobacillus reuteri

Abstract

Previously, we have shown that the glucansucrase GtfA-ΔN enzyme of Lactobacillus reuteri 121, incubated with sucrose, efficiently glucosylated catechol and we structurally characterized catechol glucosides with up to five glucosyl units attached (te Poele et al. in Bioconjug Chem 27:937-946, 2016). In the present study, we observed that upon prolonged incubation of GtfA-ΔN with 50 mM catechol and 1000 mM sucrose, all catechol had become completely glucosylated and then started to reappear. Following depletion of sucrose, this glucansucrase GtfA-ΔN used both α-D-Glcp-catechol and α-D-Glcp-(1→4)-α-D-Glcp-catechol as donor substrates and transferred a glucose unit to other catechol glycoside molecules or to sugar oligomers. In the absence of sucrose, GtfA-ΔN used α-D-Glcp-catechol both as donor and acceptor substrate to synthesize catechol glucosides with 2 to 10 glucose units attached and formed gluco-oligosaccharides up to a degree of polymerization of 4. Also two other glucansucrases tested, Gtf180-ΔN from L. reuteri 180 and GtfML1-ΔN from L. reuteri ML1, used α-D-Glcp-catechol and di-glucosyl-catechol as donor/acceptor substrate to synthesize both catechol glucosides and gluco-oligosaccharides. With sucrose as donor substrate, the three glucansucrase enzymes also efficiently glucosylated the phenolic compounds pyrogallol, resorcinol, and ethyl gallate; also these mono-glucosides were used as donor/acceptor substrates.

Keywords: Acceptor reaction; Catechol glucosides; Glucansucrase; Glucosyl donor; Lactobacillus reuteri.

Fig. 1

NP-HPLC product profiles (276 nm) of an incubation of 0.5 mg/mL GtfA-ΔN with 50 mM catechol and 1000 mM sucrose, incubated for 0, 30, 60, 80, 105, 135, and 240 min. Asterisk catechol peak at 4 min; double asterisks catG1 peak at 13.3 min in the gray area; triple asterisks cat⁴G2 and cat⁶G2 peaks at 17.3 and 18.7 min, respectively

Fig. 2

Graph showing the sucrose (filled circle) conversion and fructose (filled square) release during incubation of 0.5 mg/mL GtfA-ΔN with 50 mM catechol and 1000 mM sucrose for 240 min. No significant glucose (filled triangle) release was detected. The data represent the means of three independent enzymatic carbohydrate detection assays. Error bars are ±1 standard deviation

Fig. 3

NP-HPLC product profiles (276 nm) of an incubation of 0.25 mg/mL GtfA-ΔN with 50 mM catG1 for t = 0, 30, and 60 min without sucrose and at t = 70, 90, and 120 min after the addition of 200 mM sucrose at 60 min. Asterisk catechol; double asterisks catG1 (in the gray area); triple asterisks catG2

Fig. 4

Effects of glucosyl donor substrate concentration, sucrose (filled triangle) and catG1 (filled circle), on initial GtfA-ΔN enzyme activity in reaction buffer and 0.125 mg/mL GtfA-ΔN at 37 °C and pH 4.7. Error bars are ±1 standard deviation

Fig. 5

One-dimensional ¹H NMR spectra of catechol glucosides produced by a GtfA-ΔN with 100 mM catG1 and 1000 mM sucrose, b GtfA-ΔN with 100 mM catG1, c GtfA-ΔN with cat⁴G2, d Gtf180-ΔN with cat³G2, and e Gtf180-ΔN with cat⁶G2. Peaks are marked with structural-reporter-group signals derived from te Poele et al. (2016) and Devlamynck et al. (2016), shown in Fig. 6

Fig. 6

Overview of structural-reporter-group signals derived from te Poele et al. (2016) and Devlamynck et al. (2016) used for interpretation of 1D ¹H NMR spectra

Fig. 7

Effects of the glucosidic linkage type of di-glucosylated catechol on the initial enzyme activity in the reaction buffer and 1.25 mg/mL GtfA-ΔN, Gtf180-ΔN, and GtfMLI-ΔN at 37 °C and pH 4.7. The data represent the means of two independent enzyme activity assays; cat³G2 (white bar); cat⁴G2 (black bar); cat⁶G2 (grey bar). Error bars are ±1 standard deviation