A transglycosylating 1,3(4)-beta-glucanase from rhodothermus marinus NMR analysis of enzyme reactions.
The enzymatic hydrolysis of polysaccharides by the 1, 3(4)-beta-glucanase (LamR) from Rhodothermus marinus has been explored. The enzyme cleaves the 1,3-beta-linkages of 3-O-substituted glucose units in 1,3-beta-glucans such as laminarin and curdlan, and also the 1,4-beta-linkages of 3-O-substituted beta-glucose in beta-glucans such as lichenin and 1,3-1, 4-beta-glucan from the cell walls of barley endosperm. The polysaccharide substrates (laminarin, curdlan and barley beta-glucan) were characterised using NMR spectroscopy. The reaction of LamR with its substrates was followed by recording one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectra at suitable time intervals after addition of the enzyme. It is shown that hydrolysis occurs with retention of the anomeric configuration and that LamR performs transglycosylation to generate both 1, 3-beta-glycosidic and 1,4-beta glycosidic linkages. The transglycosylation results in, e.g. formation of the trisaccharide 4-O-glucosyl-laminaribiose from exclusively 1,3-beta-oligoglucosides. When barley 1,3-1,4-beta-glucan was incubated with LamR the beta-1, 4-linkages of 3-O-substituted beta-glycosyl residues were rapidly hydrolysed. Simultaneously de novo formation of 1,3-beta-glycosidic linkages was observed which, however, were cleaved during prolonged incubations. It is shown that a laminaribiosyl unit is the minimum requirement for formation of an enzyme-substrate complex and subsequent hydrolysis/transglycosylation.
Petersen
B O
BO
Department of Chemistry, Carlsberg Laboratory, Valby, Denmark.
Krah
M
M
Duus
J O
JO
Thomsen
K K
KK
D002236:Carbohydrate Conformation; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000737:chemistry / Q000378:metabolism*; D005936:Glucans / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D001467:Hordeum / Q000737:chemistry; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D047071:beta-Glucans
D005936:Glucans; D011134:Polysaccharides; D047071:beta-Glucans; C038459:curdlan; C008247:laminaran; D004693:Glucan Endo-1,3-beta-D-Glucosidase