Structural determinants of alternating (α1 → 4) and (α1 → 6) linkage specificity in reuteransucrase of Lactobacillus reuteri

Abstract

The glucansucrase GTFA of Lactobacillus reuteri 121 produces an α-glucan (reuteran) with a large amount of alternating (α1 → 4) and (α1 → 6) linkages. The mechanism of alternating linkage formation by this reuteransucrase has remained unclear. GTFO of the probiotic bacterium Lactobacillus reuteri ATCC 55730 shows a high sequence similarity (80%) with GTFA of L. reuteri 121; it also synthesizes an α-glucan with (α1 → 4) and (α1 → 6) linkages, but with a clearly different ratio compared to GTFA. In the present study, we show that residues in loop977 (970DGKGYKGA977) and helix α4 (1083VSLKGA1088) are main determinants for the linkage specificity difference between GTFO and GTFA, and hence are important for the synthesis of alternating (α1 → 4) and (α1 → 6) linkages in GTFA. More remote acceptor substrate binding sites (i.e.+3) are also involved in the determination of alternating linkage synthesis, as shown by structural analysis of the oligosaccharides produced using panose and maltotriose as acceptor substrate. Our data show that the amino acid residues at acceptor substrate binding sites (+1, +2, +3…) together form a distinct physicochemical micro-environment that determines the alternating (α1 → 4) and (α1 → 6) linkages synthesis in GTFA.

Structural determinants in a glucose-containing lipopolysaccharide from Mycobacterium tuberculosis critical for inducing a subset of protective T cells

“…The protons between δ 3.82 and 3.67 ppm with the corresponding 13 C chemical shift at δ 62.5 ppm was attributed to the H-6/C-6 β-Glc p units ( 24 ). The 13 C at δ 66.0 ppm showing two different proton chemical shifts at δ 3.84 and δ 3.63 ppm is possibly due to the diastereotopic relationship and was attributed to the H-6/C-6 of the α-(1→6)-Glc p -(1→2)-glyceric acid unit ( 25 , 26 ). We could assign the 13 C of the methylene peaks at δ 63.8 ppm with the proton chemical shifts between δ4.42 and 4.22 ppm to the H-6/C-6 of the Glc p units that are acyl-substituted.…”

Interactions of Surface Exopolysaccharides From Bifidobacterium and Lactobacillus Within the Intestinal Environment

“…According to the chemical composition and the way of synthesis two types of EPS, homopolysaccharides (HoPS) and heteropolysaccharides (HePS), are synthesized by LAB or bifidobacteria (Hidalgo-Cantabrana et al, 2014 ; Torino et al, 2015 ; Caggianiello et al, 2016 ; Zannini et al, 2016 ). In short, HoPS are composed of glucose or fructose and, depending on the position of the carbon involved in the linkage, they could be α-glucans, β-glucans, or β-fructans (Figure 2 ) (Kralj et al, 2004 ; Anwar et al, 2008 ; Sims et al, 2011 ; Meng et al, 2016b ; Nácher-Vázqueza et al, 2017 ; Dertli et al, 2018 ). Sucrose is the donor substrate for glycosyl hydrolase (GH) enzymes, named glucansucrases (family GH70), and fructansucrases (GH68) that catalyze the synthesis of α-glucans or β-fructans, respectively, and are located extracellularly (van Hijum et al, 2006 ; Meng et al, 2016a ; Gangoiti et al, 2018 ; Yan et al, 2018 ).…”

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

“…Residues surrounding the glucansucrase active site have been subjected to several mutagenesis studies aiming to identify the structural determinants of product size and linkage specificity in these enzymes. ,,,,,, The three residues (N1029, W1065, and Q1140) targeted in this work have been studied before, but in reactions with different acceptor substrates. Notably, all three residues are fully conserved within glucansucrases, and they play an important role in the transglycosylation reaction.…”