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.
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.
From the Departments of Microbial Physiology and.
Xiangfeng
X
Meng
Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
Tjaard
T
Pijning
From the Departments of Microbial Physiology and.
Justyna M
JM
Dobruchowska
From the Departments of Microbial Physiology and.
Gerrit J
GJ
Gerwig
From the Departments of Microbial Physiology and l.dijkhuizen@rug.nl.
Lubbert
L
Dijkhuizen
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D008401:Gas Chromatography-Mass Spectrometry; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052200:Limosilactobacillus reuteri / Q000201:enzymology*; D008745:Methylation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D066244:Proton Magnetic Resonance Spectroscopy; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D016695:Glycosyltransferases; C033837:alternansucrase
GTF180; carbohydrate; enzyme mutation; glucansucrase; polysaccharide; product specificity; protein engineering; site-directed mutagenesis; α-glucan