Mutational Analysis of the Role of the Glucansucrase Gtf180-ΔN Active Site Residues in Product and Linkage Specificity with Lactose as Acceptor Substrate

Abstract

Glucansucrase Gtf180-ΔN from Lactobacillus reuteri uses lactose as acceptor substrate to synthesize five glucosylated lactose molecules (F1-F5) with a degree of polymerization (DP) of 3-4 (GL34) and with (α1→2)/(α1→3)/(α1→4) glycosidic linkages. Q1140/W1065/N1029 mutations significantly changed the GL34 product ratios. Q1140 mutations clearly decreased F3 3'-glc-lac with an (α1→3) linkage and increased F4 4',2-glc-lac with (α1→4)/(α1→2) linkages. Formation of F2 2-glc-lac with an (α1→2) linkage and F4 was negatively affected in most W1065 and N1029 mutants, respectively. Mutant N1029G synthesized four new products with additional (α1→3)-linked glucosyl moieties (2xDP4 and 2xDP5). Sucrose/lactose strongly reduced Gtf180-ΔN hydrolytic activity and increased transferase activity of Gtf180-ΔN and mutant N1029G, in comparison to activity with sucrose alone. N1029/W1065/Q1140 thus are key determinants of Gtf180-ΔN linkage and product specificity in the acceptor reaction with lactose. Mutagenesis of key residues in Gtf180-ΔN may allow synthesis of tailor-made mixtures of novel lactose-derived oligosaccharides with potential applications as prebiotic compounds in food/feed and in pharmacy/medicine.

Keywords: Lactobacillus reuteri; glucansucrase; lactose; oligosaccharides; prebiotics; protein engineering; transglycosylation.

Scheme 1. Structures of F1–F5 and G1–G4

Red arrows reflect possible elongation of the corresponding compounds from the mixture GL34, F2–F5, to form G1–G4.

Figure 1

Views of the acceptor substrate lactose (yellow carbon atoms) docked in a glucosyl-enzyme intermediate constructed using the crystal structure of L. reuteri 180 Gtf180-ΔN (PDB 3KLK(18)). Three different poses are shown representing (a) (α1→2) transglycosylation at the reducing end, (b) (α1→3) transglycosylation at the nonreducing end, and (c) (α1→4) transglycosylation at the nonreducing end. Hydrogen bonds between amino acid residues and lactose are shown as red dotted lines; the arrow indicates the relevant hydroxyl group of lactose to attack the C1 atom of the glucosyl-enzyme intermediate (indicated with an asterisk).

Figure 2

Effects of mutations in residues N1029, W1065, and Q1140 on the synthesis of structures F1–F5 in the GL34 mixtures, relative to wild-type Gtf180-ΔN (100%). Reactions were carried out with 0.5 M sucrose and 0.3 M lactose, catalyzed by 1 U mL^–1 of these enzymes at 37 °C for 24 h. The experiments were carried out in duplicate.

Figure 3

HPAEC-PAD profiles of lactose-derived oligosaccharide products in incubation mixtures with (a) Gtf180-ΔN wild type and corresponding mutants (b) N1029G and (c) W1065M using 1 U mL^–1 (total activity) at 37 °C in 25 mM sodium acetate buffer (pH 4.7) with 1 mM CaCl₂ for 24 h. The red arrows indicate new peaks in the reaction mixture catalyzed by mutants of Gtf180-ΔN, identified as G1–G4.

Figure 4

Influence of sucrose concentration on initial activities (hydrolytic, transferase, and total activity) of different enzymes: (1) Gtf180-ΔN; (2) W1065M, and (3) N1029G when using (a) sucrose as donor substrate and 200 mM lactose as acceptor substrate or (b) only sucrose as substrate, with 5 μg of the protein of the corresponding enzymes at 37 °C in 25 mM sodium acetate/1 mM CaCl₂ buffer, pH 4.7. Experiments were carried out in duplicate.

Figure 5

500 MHz 1D ¹H NMR spectra of G1–G4 fractions from the reaction mixture with Gtf180-ΔN N1029G (see Figure S1), recorded at 25 °C in D₂O. Anomeric signals of each fraction were labeled according to the legends of corresponding structures indicated in Scheme 1.