Characterization of the Functional Roles of Amino Acid Residues in Acceptor-binding Subsite +1 in the Active Site of the Glucansucrase GTF180 from Lactobacillus reuteri 180

Abstract

␣-Glucans produced by glucansucrase enzymes hold strong potential for industrial applications. The exact determinants of the linkage specificity of glucansucrase enzymes have remained largely unknown, even with the recent elucidation of glucansucrase crystal structures. Guided by the crystal structure of glucansucrase GTF180-⌬N from Lactobacillus reuteri 180 in complex with the acceptor substrate maltose, we identified several residues (Asp-1028 and Asn-1029 from domain A, as well as Leu-938, Ala-978, and Leu-981 from domain B) near subsite ؉1 that may be critical for linkage specificity determination, and we investigated these by random site-directed mutagenesis. First, mutants of Ala-978 (to Leu, Pro, Phe, or Tyr) and Asp-1028 (to Tyr or Trp) with larger side chains showed reduced degrees of branching, likely due to the steric hindrance by these bulky residues. Second, Leu-938 mutants (except L938F) and Asp-1028 mutants showed altered linkage specificity, mostly with increased (␣136) linkage synthesis. Third, mutation of Leu-981 and Asn-1029 significantly affected the transglycosylation reaction, indicating their essential roles in acceptor substrate binding. In conclusion, glucansucrase product specificity is determined by an interplay of domain A and B residues surrounding the acceptor substrate binding groove. Residues surrounding the ؉1 subsite thus are critical for activity and specificity of the GTF180 enzyme and play different roles in the enzyme functions. This study provides novel insights into the structurefunction relationships of glucansucrase enzymes and clearly shows the potential of enzyme engineering to produce tailormade ␣-glucans.

4,3-α-Glucanotransferase, a novel reaction specificity in glycoside hydrolase family 70 and clan GH-H

“…A second difference is Asp991 of L. fermentum NCC 2970 GtfB; it replaces the conserved Asn present in GSs and most GtfB-like 4,6-α-GTases. In the GS of L. reuteri 180 this residue was found to be essential for activity and linkage specificity31. The preceding Asp is conserved in L. fermentum GtfB (Asp 990).…”

“…However, the lack of 3D structural information and of α1 → 2 and α1 → 3 synthesizing maltodextrins/starch utilizing type of GH70 enzymes has limited our understanding of the structural features determining this different substrate and product specificity. For GSs, the crystal structure of GTF180-ΔN with a bound maltose acceptor combined with site-directed mutagenesis experiments revealed that the linkage specificity is controlled by residues forming the acceptor substrate binding subsites +1/+22029303132. Despite their different domain organization, GtfB, GtfC, and GtfD 4,6-α-GTase enzymes display high conservation in their motifs I to IV, particularly in residues forming the acceptor substrate binding subsites in GSs2326.…”

Processivity of dextransucrases synthesizing very-high-molar-mass dextran is mediated by sugar-binding pockets in domain V

“…For that purpose, a better understanding of the dextransucrase structural determinants dictating polymer elongation appears necessary. In the last decades, sequence analyses and X-Ray 3D-structure characterizations allowed pointing some amino acid residues involved in linkage specificity [6][7][8][9][10][11]. However, less is known on the enzyme structural determinants controlling the polymer size.…”

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

“…Residues in homology regions I-IV in domain A are critical for linkage specificity determination of glucansucrase enzymes, as shown in previous mutagenesis studies 15 39 40 41 42 43 . Moreover, residues from domain B have been shown to shape the acceptor binding sites and hence determine the linkage specificity 10 16 41 44 45 . The amino acid sequences of these regions in GTFO and GTFA were compared by alignment with the other glucansucrase enzymes from LAB ( Fig.…”