Glucosylation of Catechol with the GTFA Glucansucrase Enzyme from Lactobacillus reuteri and Sucrose as Donor Substrate

Abstract

Lactic acid bacteria use glucansucrase enzymes for synthesis of gluco-oligosaccharides and polysaccharides (α-glucans) from sucrose. Depending on the glucansucrase enzyme, specific α-glucosidic linkages are introduced. GTFA-ΔN (N-terminally truncated glucosyltransferase A) is a glucansucrase enzyme of Lactobacillus reuteri 121 that synthesizes the reuteran polysaccharide with (α1 → 4) and (α1 → 6) glycosidic linkages. Glucansucrases also catalyze glucosylation of various alternative acceptor substrates. At present it is unclear whether the linkage specificity of these enzymes is the same in oligo/polysaccharide synthesis and in glucosylation of alternative acceptor substrates. Our results show that GTFA-ΔN glucosylates catechol into products with up to at least 5 glucosyl units attached. These catechol glucosides were isolated and structurally characterized using 1D/2D (1)H NMR spectroscopy. They contained 1 to 5 glucose units with different (α1 → 4) and (α1 → 6) glycosidic linkage combinations. Interestingly, a branched catechol glucoside was also formed along with a catechol glucoside with 2 successive (α1 → 6) glycosidic linkages, products that are absent when only sucrose is used as both glycosyl donor and acceptor substrate.

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

“…α- d -Glc p -catechol (catG1) and α- d -Glc p -(1→4)-α- d -Glc p -catechol (cat 4 G2) synthesized by GtfA-ΔN (te Poele et al 2016), and α- d -Glc p -(1→3)-α- d -Glc p -catechol (cat 3 G2) and α- d -Glc p -(1→6)-α- d -Glc p -catechol (cat 6 G2) synthesized by Gtf180-ΔN (Devlamynck et al 2016), were isolated and purified using preparative normal-phase HPLC (NP-HPLC) as described previously (te Poele et al 2016). GtfA-ΔN enzyme activity on catG1 was measured at six different catG1 concentrations ranging from 3.1 to 100 mM using 0.125 mg/mL GtfA-ΔN.…”

“…Glucansucrase enzymes are interesting biocatalysts for the glycosylation of small organic molecules, because they are promiscuous towards a wide range of acceptor substrates (Monsan et al 2010; Leemhuis et al 2012). Using inexpensive sucrose as glycosyl donor, glucansucrase enzymes glucosylate, for instance, phenolic compounds such as catechol (Meulenbeld and Hartmans 2000; Devlamynck et al 2016; te Poele et al 2016) and hydroquinone (Seo et al 2009; Devlamynck et al 2016), primary alcohols (Seibel et al 2006; Devlamynck et al 2016), and l -ascorbic acid (Kim et al 2010). Glycosylation may change the physico-chemical and biological properties of molecules, enhance bioavailability of antibiotics, or improve stability of molecules against auto-oxidation (Desmet et al 2012).…”

“…Glycosylation may change the physico-chemical and biological properties of molecules, enhance bioavailability of antibiotics, or improve stability of molecules against auto-oxidation (Desmet et al 2012). Recently, we characterized a series of catechol glucosides produced by GtfA-ΔN, after incubation with catechol and sucrose (te Poele et al 2016). Catechol glucosides up to a degree of polymerization (DP) of 5, with different combinations of (α1→4) and (α1→6) linkages, were structurally characterized using 1D/2D 1 H NMR spectroscopy, and compared to α-glucan structures synthesized by GtfA-ΔN from sucrose only.…”

“…Interesting similarities were found, but also clear structural differences. A branched catechol glucoside was identified and a catechol glucoside with two successive (α1→6) glycosidic linkages, structures that were not found when GtfA-ΔN was incubated with sucrose only (te Poele et al 2016). …”

“…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.…”