Mutations in Amino Acid Residues of Limosilactobacillus reuteri 121 GtfB 4,6-α-Glucanotransferase that Affect Reaction and Product Specificity

Yawen Jiang, Xiaoxiao Li, Tjaard Pijning, Yuxiang Bai, Lubbert Dijkhuizen

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Limosilactobacillus reuteri 121 4,6-α-glucanotransferase (Lr121 4,6-α-GTase), belonging to the glycosyl hydrolase (GH) 70 GtfB subfamily, converts starch and maltodextrins into linear isomalto/malto polysaccharides (IMMPs) with consecutive (α1 → 6) linkages. The recent elucidation of its crystal structure allowed identification and analysis of further structural features that determine its reaction and product specificity. Herein, sequence alignments between GtfB enzymes with different product linkage specificities (4,6-α-GTase and 4,3-α-GTase) identified amino acid residues in GH70 homology motifs, which may be critical for reaction and product specificity. Based on these alignments, four Lr121 GtfB-ΔN mutants (I1020M, S1057P, H1056G, and Q1126I) were constructed. Compared to wild-type Lr121 GtfB-ΔN, mutants S1057P and Q1126I had considerably improved catalytic efficiencies. Mutants H1056G and Q1126I showed a 9% decrease and an 11% increase, respectively, in the ratio of (α1 → 6) over (α1 → 4) linkages in maltodextrin-derived products. A change in linkage type (e.g., (α1 → 6) linkages to (α1 → 3) linkages) was not observed. The possible functional roles of these Lr121 GtfB-ΔN residues located around the acceptor substrate-binding subsites are discussed. The results provide new insights into structural determinants of the reaction and product specificity of Lr121 GtfB 4,6-α-GTase.

Original languageEnglish
Article number1c07618
Pages (from-to)1952-1961
Number of pages10
JournalJournal of Agricultural and Food Chemistry
Volume70
Issue number6
Early online date7-Feb-2022
DOIs
Publication statusPublished - 16-Feb-2022

Keywords

  • α-glucanotransferase
  • site-directed mutagenesis
  • catalytic mechanism
  • reaction and product specificity

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