Construction of chimeric glucansucrases for analyzing substrate-binding regions that affect the structure of glucan products.
A gene that encodes dextransucrase S (dsrS) from Leuconostoc mesenteroides NRRL B-512F encodes a glucansucrase dextransucrase S (DSRS) which mainly produces water-soluble glucan (dextran), while the dsrT5 gene derived from dsrT of the B-512F strain encodes an enzyme dextransucrase T5 (DSRT5), which mainly produces water-insoluble glucan. Tyr340-Asn510 of DSRS and Tyr307-Asn477 of DSRT5 (Site 1), Lys696-Gly768 of DSRS and Lys668-Gly740 of DSRT5 (Site 2), and Asn917-Lys1131 of DSRS and Asn904-Lys1118 of DSRT5 (Site 3) were exchanged and six different chimeric enzymes were constructed. Water-soluble glucan produced by recombinant DSRS was composed of 64% 6-linked glucopyranoside (Glcp), 9% 3,6-linked Glcp, and 13% 4-linked Glcp. Water-insoluble glucan produced by recombinant DSRT5 was composed of 47% 6-linked Glcp and 43% 3-linked Glcp. All of the chimeric enzymes produced glucans different from the ones produced by their parental enzymes. Some of the glucans produced by chimeric enzymes were extremely changed. The Site 1 chimeric enzyme of DSRS (STS1) produced water-soluble glucan composed mostly of 6-linked Glcp. That of DSRT5 (TST1) produced water-insoluble glucan composed mostly of 4-linked Glcp. The Site 3 chimeric enzyme of DSRS (STS3) produced mainly water-insoluble glucan, DSRT5 (TST3) produced mainly water-soluble glucans, and all of the glucan fractions consisted of 3-Glcp, 4-Glcp, and 6-Glcp. The amounts of the three linkages in the water-soluble glucan produced by TST3 were about 1:1:1. Site 1 was assumed to be important for making or avoiding making alpha-1,4 linkages, while Site 3 was assumed to be important for determining the kinds of glucosyl linkages made.
Funane
Kazumi
K
National Food Research Institute, Tsukuba, Ibaraki, Japan. funane@nfri.affrc.go.jp
Ishii
Tadashi
T
Terasawa
Kazue
K
Yamamoto
Tomoko
T
Kobayashi
Mikihiko
M
D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D020134:Catalytic Domain; D005936:Glucans / Q000378:metabolism*; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007934:Leuconostoc / Q000201:enzymology; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis*; D012995:Solubility; D013210:Staphylococcus / Q000201:enzymology; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005936:Glucans; D011993:Recombinant Fusion Proteins; D016695:Glycosyltransferases; C033837:alternansucrase