Hybrid reuteransucrase enzymes reveal regions important for glucosidic linkage specificity and the transglucosylation/hydrolysis ratio.
The reuteransucrase enzymes of Lactobacillus reuteri strain 121 (GTFA) and L. reuteri strain ATCC 55730 (GTFO) convert sucrose into alpha-d-glucans (labelled reuterans) with mainly alpha-(1-->4) glucosidic linkages (50% and 70%, respectively), plus alpha-(1-->6) linkages. In the present study, we report a detailed analysis of various hybrid GTFA/O enzymes, resulting in the identification of specific regions in the N-termini of the catalytic domains of these proteins as the main determinants of glucosidic linkage specificity. These regions were divided into three equal parts (A1-3; O1-3), and used to construct six additional GTFA/O hybrids. All hybrid enzymes were able to synthesize alpha-glucans from sucrose, and oligosaccharides from sucrose plus maltose or isomaltose as acceptor substrates. Interestingly, not only the A2/O2 regions, with the three catalytic residues, affect glucosidic linkage specificity, but also the upstream A1/O1 regions make a strong contribution. Some GTFO derived hybrid/mutant enzymes displayed strongly increased transglucosylation/hydrolysis activity ratios. The reduced sucrose hydrolysis allowed the much improved conversion of sucrose into oligo- and polysaccharide products. Thus, the glucosidic linkage specificity and transglucosylation/hydrolysis ratios of reuteransucrase enzymes can be manipulated in a relatively simple manner. This engineering approach has yielded clear changes in oligosaccharide product profiles, as well as a range of novel reuteran products differing in alpha-(1-->4) and alpha-(1-->6) linkage ratios.
Kralj
Slavko
S
Centre for Carbohydrate Bioprocessing, TNO-University of Groningen, Haren, The Netherlands.
van Leeuwen
Sander S
SS
Valk
Vincent
V
Eeuwema
Wieger
W
Kamerling
Johannis P
JP
Dijkhuizen
Lubbert
L
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002236:Carbohydrate Conformation; D020134:Catalytic Domain; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D005936:Glucans / Q000096:biosynthesis; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D007534:Isomaltose / Q000737:chemistry / Q000378:metabolism; D052200:Limosilactobacillus reuteri / Q000145:classification / Q000201:enzymology*; D008320:Maltose / Q000737:chemistry / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D013379:Substrate Specificity; D013395:Sucrose / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D004187:Disaccharides; D005936:Glucans; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D013395:Sucrose; D007534:Isomaltose; D008320:Maltose; D005964:Glucosyltransferases; C060525:leucrose