Characterization of the (Engineered) Branching Sucrase GtfZ-CD2 from Apilactobacillus kunkeei for Efficient Glucosylation of Benzenediol Compounds

Abstract

Branching sucrases, a subfamily of Glycoside Hydrolase family (GH70), display transglycosidase activity using sucrose as donor substrate to catalyze glucosylation reaction in the presence of suitable acceptor substrates. In this study, the (α1→3) branching sucrase GtfZ-CD2 from Apilactobacillus kunkeei DSM 12361 was demonstrated to glucosylate benzenediol compounds (i.e., catechol, resorcinol, and hydroquinone) to form monoglucoside and diglucoside products. The production and yield of catechol glucosylated products were significantly higher than that of resorcinol and hydroquinone, revealing a preference for adjacent aromatic hydroxyl groups in glucosylation. Amino residues around acceptor substrate binding subsite +1 were targeted for semirational mutagenesis, yielding GtfZ-CD2 variants with improved resorcinol and hydroquinone glucosylation. Mutant L1560Y with improved hydroquinone mono-glucosylated product synthesis allowed enzymatic conversion of hydroquinone into α-arbutin. This study thus revealed the high potential of GH70 branching sucrases for glucosylating noncarbohydrate molecules. IMPORTANCE Glycosylation represents one of the most important ways to expand the diversity of natural products and improve their physico-chemical properties. Aromatic polyphenol compounds widely found in plants are reported to exhibit various remarkable biological activities; however, they generally suffer from low solubility and stability, which can be improved by glycosylation. Our present study on the glucosylation of benzenediol compounds by GH70 branching sucrase GtfZ-CD2 and its semirational engineering to improve the glucosylation efficiency provides insight into the mechanism of acceptor substrates binding and its glucosylation selectivity. The results demonstrate the potential of using branching sucrase as an effective enzymatic glucosylation tool.

Keywords: GH70; benzenediol; branching sucrase; glucosylation; α-arbutin.

FIG 1

HPLC analysis of glucosylated products produced by incubating GtfZ-CD2 with benzenediol compounds. GtfZ-CD2 (0.5 U/mL) was incubated with 150 mM sucrose as donor substrate, and 150 mM catechol (A), resorcinol (B), and hydroquinone (C), respectively, as the acceptor substrate in 50 sodium acetate buffer pH 5.5, 1 mM CaCl₂ at 30°C for 120 h. S: sucrose; F: fructose; G: glucose; Cat: catechol; Res: resorcinol; Hq: hydroquinone. The monoglucoside and diglucoside products of catechol, resorcinol and hydroquinone were labeled as corresponding G1 and G2.

FIG 2

¹H NMR spectra and structures of benzenediol (catechol, resorcinol, and hydroquinone) glucosylated products, i.e., Cat-G1, Cat-G2, Res-G1, Res-G2, Hq-G1, Hq-G2.

FIG 3

Effects of different initial concentrations of benzenediol on the production (mM) and yield (%) of glucosylated products by GtfZ-CD2. A: catechol; B: resorcinol; C: hydroquinone. Values are the mean of three replicate experiments and error bars represent the Standard Deviation (SD) of these replicates.

FIG 4

Molecular docking of catechol (A), resorcinol (B) and hydroquinone (C) in the acceptor substrate binding site +1 of GtfZ-CD2. For each ligand, two different productive poses are shown. Amino acid residues within 5 Å of each ligand are viewed as sticks. Amino acid residues from domain A of GtfZ-CD2 are depicted in blue while residues from domain B are depicted in green. The covalent glucosyl intermediate on D1634 is shown with yellow sticks.

FIG 5

Analysis of the synthesis (mM), yield (%) and distribution of glucosylated products formed by GtfZ-CD2 and constructed mutant enzymes with catechol (A), resorcinol (B) and hydroquinone (C) as acceptor substrates. Wild-type and mutant enzymes (0.5 U/mL) were incubated with 200 mM acceptor substrates and 1 M sucrose in 50 mM sodium acetate buffer pH 5.5, 1 mM CaCl₂ at 30°C for 120 h. The glucosylated products were quantified by HPLC using their respective standards. Values are the mean of three replicate experiments and error bars represent the Standard Deviation (SD) of these replicates.