Glycosynthase activity of hybrid aspen xyloglucan endo-transglycosylase PttXET16-34 nucleophile mutants.
Glycosynthases are active-site mutants of glycoside hydrolases that catalyse glycosyl transfer using suitable activated donor substrates without competing product hydrolysis (S. M. Hancock, M. D. Vaughan and S. G. Withers, Curr. Opin. Chem. Biol., 2006, 10, 509-519). Site-directed mutagenesis of the catalytic nucleophile, Glu-85, of a Populus tremula x tremuloides xyloglucan endo-transglycosylase (PttXET16-34, EC 2.4.1.207) into alanine, glycine, and serine yielded enzymes with glycosynthase activity. Product analysis indicated that PttXET16-34 E85A in particular was able to catalyse regio- and stereospecific homo- and hetero-condensations of alpha-xylogluco-oligosaccharyl fluoride donors XXXGalphaF and XLLGalphaF to produce xyloglucans with regular sidechain substitution patterns. This substrate promiscuity contrasts that of the Humicola insolens Cel7B E197A glycosynthase, which was not able to polymerise the di-galactosylated substrate XLLGalphaF. The production of the PttXET16-34 E85A xyloglucosynthase thus expands the repertoire of glycosynthases to include those capable of synthesising structurally homogenenous xyloglucans for applications.
Piens
Kathleen
K
School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, 106 91, Stockholm, Sweden.
Henriksson
Anna-Maria
AM
Gullfot
Fredrika
F
Lopez
Marie
M
Fauré
Régis
R
Ibatullin
Farid M
FM
Teeri
Tuula T
TT
Driguez
Hugues
H
Brumer
Harry
H
D001665:Binding Sites; D002384:Catalysis; D005461:Fluorine / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000378:metabolism*; D016695:Glycosyltransferases / Q000235:genetics* / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism*; D032107:Populus / Q000201:enzymology*; D021241:Spectrometry, Mass, Electrospray Ionization; D013237:Stereoisomerism; D013379:Substrate Specificity
D009844:Oligosaccharides; D005461:Fluorine; D016695:Glycosyltransferases; C073693:xyloglucan endotransglycosylase; D006026:Glycoside Hydrolases