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.
Highlights
Our results show that loop[977] (970DGKGYKGA977) and helix α4 (1083VSLKGA1088) residues are the major determinants for the linkage specificity difference between GTFO and GTFA
A are critical for linkage specificity determination of glucansucrase enzymes, as shown in previous mutagenesis studies[15,39,40,41,42,43]
The amino acid sequences of these regions in GTFO and GTFA were compared by alignment with the other glucansucrase enzymes from Lactic acid bacteria (LAB) (Fig. 1a)
Summary
GTFO-∆N mutant S1065H:S1066A synthesized polysaccharides similar to those produced by wild-type GTFO-∆N These results demonstrate that loop[977] and helix α4 play an important role in determining the linkage specificity of GTFO and GTFA, but not residues S1065 and S1066. Mutant enzymes only involving helix α4 residues produced similar amounts of panose and maltotriose, slightly lower amount of glucosyl-(α1 → 4)-panose and higher amounts of glucosyl-(α 1 → 6)-(α1 → 4)-panose, suggesting that these mutations enhanced the ability of enzymes to synthesize alternating (α1 → 6) and (α1 → 4) linkages, still retained the relative high ability to form successive (α1 → 4) linkages. Mutant enzymes containing mutations both in loop[977] and helix α4 showed both reduced amounts
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