Glycosynthase activity of hybrid aspen xyloglucan endo-transglycosylase PttXET16-34 nucleophile mutants

Abstract

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.

Mechanism-based Labeling Defines the Free Energy Change for Formation of the Covalent Glycosyl-enzyme Intermediate in a Xyloglucan endo-Transglycosylase

“…The enzyme concentration was determined from A 280 measurements (⑀ calculated ϭ 74490 M Ϫ1 cm Ϫ1 ). The PttXET16 -34 catalytic nucleophile variant E85G was produced using standard sitedirected mutagenesis techniques (24). Horse heart myoglobin was obtained from Sigma as a powder and dissolved in ultrapure water prior to dilution to 6 g/ml (0.36 M) in 5% aqueous acetonitrile containing 0.1% formic acid.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

“…Reaction mechanism (Kanazawa, et al, 2007) (Ohmae, et al, 2007) Xyloglucans MALDI Use of mutated aspen xyloglucan endotransglycosylase (Piens, et al, 2007) …”

The XTH Gene Family: An Update on Enzyme Structure, Function, and Phylogeny in Xyloglucan Remodeling

“…Confirmation of the identity of the catalytic nucleophile in GH16 XETs and XEHs comes from the observation that mutation of the corresponding Glu in both PttXET16-34 and TmNXG1 results in the complete loss of the wild-type activity; these nucleophile variants are nonetheless able to perform "glycosynthase" reactions (i.e. the resynthesis of xyloglucans from artificial donor substrates [a-fluoroglycosides of xyloglucan oligosaccharides]; Piens et al, 2007;Gullfot et al, 2009). Furthermore, mutation of Glu-85 in PttXET16-34 to Ala prevents formation of the covalent glycosyl-enzyme (Piens et al, 2008).…”