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.
Highlights
The ability of lactic acid bacteria to produce large amounts of exopolysaccharides has drawn strong attention for industrial applications in recent years [1,2,3,4]
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
Glucansucrase enzymes of lactic acid bacteria catalyze the synthesis of various ␣-glucans [5], which are used as biothickening agent in food industry, as plasma expander in medicine, and as separation matrix in research [1,2,3,4, 6]
Summary
The ability of lactic acid bacteria to produce large amounts of exopolysaccharides has drawn strong attention for industrial applications in recent years [1,2,3,4]. Mutations in residues Ser-1137–Asp-1141 (GTF180 numbering) following the transition state stabilizer (Asp-1136) in homology region IV (Fig. 1) have been shown to change the linkage compositions of synthesized ␣-glucan products in several glucansucrase enzymes (9, 22, 24 –28). A direct hydrogen bond with the ϩ2 C1 hydroxyl group [12] These observations confirmed the involvement of these residues in forming acceptor binding sites as predicted in previous studies and explain the altered linkage specificity caused by mutating these residues. In a recent study of dextransucrase DSRS from L. mesenteroides NRRL B-512F, guided by the homologous GTF180-⌬N crystal structure, several residues (including residues corresponding to residues Leu-938, Ala978, and Asn-1029 of GTF180) were targeted for combinatorial mutagenesis [31] Several of these mutants were found to display an altered product linkage distribution. A large number of mutants were characterized, and their ␣-glucan polysaccharide products were structurally analyzed, determining the linkage type distributions and their substitution pattern by NMR
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