Characterization of the glucansucrase GTF180 W1065 mutant enzymes producing polysaccharides and oligosaccharides with altered linkage composition.
Exopolysaccharides produced by lactic acid bacteria are extensively used for food applications. Glucansucrase enzymes of lactic acid bacteria use sucrose to catalyze the synthesis of α-glucans with different linkage compositions, size and physico-chemical properties. Crystallographic studies of GTF180-ΔN show that at the acceptor binding sites +1 and +2, residue W1065 provides stacking interactions to the glucosyl moiety. However, the detailed functional roles of W1065 have not been elucidated. We performed random mutagenesis targeting residue W1065 of GTF180-ΔN, resulting in the generation of 10 mutant enzymes that were characterized regarding activity and product specificity. Characterization of mutant enzymes showed that residue W1065 is critical for the activity of GTF180-ΔN. Using sucrose, and sucrose (donor) plus maltose (acceptor) as substrates, the mutant enzymes synthesized polysaccharides and oligosaccharides with changed linkage composition. The stacking interaction of an aromatic residue at position 1065 is essential for polysaccharide synthesis.
Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. Electronic address: x.meng@rug.nl.
Xiangfeng
X
Meng
Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. Electronic address: t.pijning@rug.nl.
Tjaard
T
Pijning
Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. Electronic address: m.tietema@knnbioplastic.nl.
Martin
M
Tietema
Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. Electronic address: judobru@uga.edu.
Justyna M
JM
Dobruchowska
Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. Electronic address: h.yin@rug.nl.
Huifang
H
Yin
Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. Electronic address: g.gerwig@rug.nl.
Gerrit J
GJ
Gerwig
Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. Electronic address: slavko.kralj@dupont.com.
Slavko
S
Kralj
Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. Electronic address: l.dijkhuizen@rug.nl.
Lubbert
L
Dijkhuizen
D008040:Genetic Linkage / Q000235:genetics*; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics*; D052200:Limosilactobacillus reuteri / Q000201:enzymology / Q000235:genetics; D008320:Maltose / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011134:Polysaccharides / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D017433:Protein Structure, Secondary; D013395:Sucrose / Q000737:chemistry / Q000378:metabolism
D009844:Oligosaccharides; D011134:Polysaccharides; D013395:Sucrose; D008320:Maltose; D016695:Glycosyltransferases; C033837:alternansucrase
GTF180; Glucansucrase; Lactic acid bacteria; Linkage specificity; W1065; α-Glucan