Identifying critical unrecognized sugar–protein interactions in GH10 xylanases from Geobacillus stearothermophilus using STD NMR

Abstract

1H solution NMR spectroscopy is used synergistically with 3D crystallographic structures to map experimentally significant hydrophobic interactions upon substrate binding in solution under thermodynamic equilibrium. Using saturation transfer difference spectroscopy (STD NMR), a comparison is made between wild‐type xylanase XT6 and its acid/base catalytic mutant E159Q – a non‐active, single‐heteroatom alteration that has been previously utilized to measure binding thermodynamics across a series of xylooligosaccharide–xylanase complexes [Zolotnitsky et al. (2004) Proc Natl Acad Sci USA 101, 11275–11280). In this study, performing STD NMR of one substrate screens binding interactions to two proteins, avoiding many disadvantages inherent to the technique and clearly revealing subtle changes in binding induced upon mutation of the catalytic Glu. To visualize and compare the binding epitopes of xylobiose–xylanase complexes, a ‘SASSY’ plot (saturation difference transfer spectroscopy) is used. Two extraordinarily strong, but previously unrecognized, non‐covalent interactions with H2–5 of xylobiose were observed in the wild‐type enzyme but not in the E159Q mutant. Based on the crystal structure, these interactions were assigned to tryptophan residues at the −1 subsite. The mutant selectively binds only the β–xylobiose anomer. The 1H solution NMR spectrum of a xylotriose–E159Q complex displays non‐uniform broadening of the NMR signals. Differential broadening provides a unique subsite assignment tool based on structural knowledge of face‐to‐face stacking with a conserved tyrosine residue at the +1 subsite. The results obtained herein by substrate‐observed NMR spectroscopy are discussed further in terms of methodological contributions and mechanistic understanding of substrate‐binding adjustments upon a charge change in the E159Q construct.

Downregulation of a UDP-Arabinomutase Gene in Switchgrass (Panicum virgatum L.) Results in Increased Cell Wall Lignin While Reducing Arabinose-Glycans

“…While a large amount of structural information can be deduced from the NMR spectra, there are regions of overlap in the anomeric signals of the xylo-oligos (Balazs et al, 2013). Specifically, the signals for monomeric α- L -Ara f side chains and the terminal α- L -Ara f in the disaccharides side chain Ara f -(1,2)-α- L -Ara f - are unresolved as are the 2-Ara f signals for D -Xyl p -(1,2)-α- L -Ara f -(1,3)- and α- L -Ara f -(1,2)-α- L -Ara f disaccharide side chains.…”

The Genus Geobacillus and Their Biotechnological Potential

“…xylanases are secreted by many strains (Balazs et al, 2013;Liu et al, 2012) and GH43 endo a-1,5-arabinanases (pectin-derived arabinan degrading) by a few (De Maayer et al, 2014;Shulami et al, 2011). p0090…”