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

α-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-ΔN 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 (α1 → 6) 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 structure-function relationships of glucansucrase enzymes and clearly shows the potential of enzyme engineering to produce tailor-made α-glucans.

Keywords: GTF180; carbohydrate; enzyme mutation; glucansucrase; polysaccharide; product specificity; protein engineering; site-directed mutagenesis; α-glucan.

FIGURE 1.

Partial alignment of the amino acid sequences of GH70 glucansucrase enzymes. Residues Leu-938, Ala-978, Leu-981, Asp-1028, and Asn-1029 of GTF180 and their corresponding residues in other glucansucrase enzymes are highlighted in blue. Two amino acid residues involved in enzyme catalysis, the nucleophile residue (NU) in homology region II and transition state stabilizer (TS) in homology region IV, are indicated.

FIGURE 2.

Stereo view of GTF180-ΔN with the acceptor maltose (yellow carbon atoms) bound in subsites +1 and +2 (PDB code 3KLL) (12). Residues Asn-1029 and Asp-1028 from domain A (blue) provide direct and indirect (water-mediated) hydrogen bonds to the non-reducing end glucosyl unit bound at subsite +1, respectively. Residues Leu-938, Leu-940, Ala-978, and Leu-981 from domain B (green) are also near subsite +1.

FIGURE 3.

a, composite model structure of EPS180 as reported in our previous study (29). The building blocks of EPS180 are presented on the right. b, 500 MHz one-dimensional ¹H NMR spectra of the α-glucans produced by GTF180-ΔN and mutants A978G, A978S, A978L, A978P, A978F, and A978Y as indicated in the figure. The H-4 signal of the terminal residue (t4, between ∼ δ 3.40 and 3.45), which is an indicator of branched linkages, is indicated.

FIGURE 4.

Methylation analysis chromatogram of polysaccharides produced by GTF180-ΔN, A978G, A978S, A978L, A978P, A978F, and A978Y as indicated in the figure by GLC-EI-MS. 1, {Glcp(1→} (1,5-di-O-acetyl-2,3,4,6-tetra-O-methylhexitol); 2, {→3)Glcp(1→)} (1,3,5-tri-O-acetyl-2,4,6-tri-O-methylhexitol); 3, {→6)Glcp(1→)} (1,5,6-tri-O-acetyl-2,3,4-tri-O-methylhexitol); 4, {→3,6)Glcp(1→)} (1,3,5,6-tetra-O-acetyl-2,4-di-O-methylhexitol).

FIGURE 5.

a, TLC analysis of products formed from incubation of GTF180-ΔN and Asn-1029 mutants (1.0 unit/ml) with 0.1 m sucrose in 25 mm NaAC, 1 mm CaCl₂ buffer, pH 4.5, at 37 °C. A mixture of glucose (G1) to maltoheptaose (G7) was used as standard (M). Lane 1, GTF180-ΔN; lane 2, N1029Y; lane 3, N1029G; lane 4, N1029T; lane 5, N1029M; and lane 6, N1029R. b, size exclusion chromatography analysis of product mixtures obtained by incubating 1.0 unit/ml GTF180-ΔN, N1029Y, and N1029M with 0.1 m sucrose in 25 mm NaAC, 1 mm CaCl₂ buffer, pH 4.5, at 37 °C. HMW, high molecular weight polysaccharides (inset enlarged for better view).

FIGURE 6.

Stereo view of docked isomaltotriose in the active site of modeled GTF180-ΔN glucosyl-enzyme intermediate (7). Residues from domain A (blue) and domain B (green) surrounding the +1 and +I′ subsites are indicated, including those (Asp-1028, Asn-1029, Leu-938, Ala-978, and Leu-981) mutated in this study.