Changes in linkage pattern of glucan products induced by substitution of Lys residues in the dextransucrase.
Dextransucrase S (DSRS) is the only active glucansucrase that has been found in Leuconostoc mesenteroides NRRL B-512F strain. Native DSRS produces mainly 6-linked glucopyranosyl residue (Glcp), while Escherichia coli recombinant DSRS was observed to produce a glucan consisting of 70% 6-linked Glcp and 15% 3,6-Glcp. Lys residues were introduced at the N-terminal end of the core domain by site-directed mutagenesis. In glucans produced by the one-point mutants T350K and S455K, the amount of 6-linked Glcp was increased to about 85% of the total glucan produced, more similar in structure to native B-512F dextran. The double mutant T350K/S455K produced adhesive, water-insoluble glucan with 77% 6-linked Glcp, 8% 3,6-linked Glcp and 4% 2,6-linked Glcp. The T350K/S455K mutant exhibited a 10-fold increase in glucosyltransferase activity over those of the parental DSRS-His(6) and its T350K and S455K mutants. This is the first report demonstrating a change in the properties of a dextransucrase or a related glucosyltransferase through simple site-directed mutagenesis to create 2,6-linked Glcp.
Funane
Kazumi
K
National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. funane@nfri.affrc.go.jp
Ishii
Tadashi
T
Ono
Hiroshi
H
Kobayashi
Mikihiko
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002236:Carbohydrate Conformation; D005936:Glucans / Q000737:chemistry* / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D016415:Sequence Alignment
D001426:Bacterial Proteins; D005936:Glucans; D005964:Glucosyltransferases; C020025:dextransucrase; D008239:Lysine