Abstract
Xylanase from Bacillus agaradhaerens belongs to a large group of glycosyl hydrolases which catalyze the degradation of xylan. The protonation behavior of titratable groups of the uniformly15N- and13C-labeled xylanase was investigated by multinuclear NMR spectroscopy. A total of 224 chemical shift titration curves corresponding to1H,13C, and15N resonances revealed pKavalues for all aspartic and glutamic acid residues, as well as for the C-terminal carboxylate and histidine residues. Most of the titratable groups exhibit a complex titration behavior, which is most likely due to the mutual interactions with other neighboring groups or due to an unusual local microenvironment. Subsite - 1 containing the catalytic dyad shows a long-range interaction over 9 Å with Asp21 via two hydrogen bonds with Asn45 as the mediator. This result illuminates the pivotal role of the conserved position 45 among family 11 endoxylanases, determining an alkaline pH optimum by asparagine residues or an acidic pH optimum by an aspartate. The asymmetric interactions of neighboring tryptophan side chains with respect to the catalytic dyad can be comprehended as a result of hydrogen bonding and aromatic stacking. Most of the chemical shift-pH profiles of the backbone amides exhibit biphasic behavior with two distinct inflection points, which correspond to the pKα values of the nearby acidic side chains. However, the alternation of both positive and negative slopes of individual amide titration curves is interpreted as a consequence of a simultaneous reorganization of side chain conformational space at pH ≈6 and/or an overall change in the hydrogen network in the substrate binding cleft.
Original language | English |
---|---|
Pages (from-to) | 5820-5831 |
Number of pages | 12 |
Journal | Biochemistry |
Volume | 43 |
Issue number | 19 |
DOIs | |
Publication status | Published - 18 May 2004 |
Keywords
- asparagine
- aspartic acid
- glutamic acid
- glycosidase
- histidine
- tryptophan
- xylan
- xylan endo 1,3 beta xylosidase
- acidity
- alkalinity
- article
- Bacillus
- Bacillus agaradherens
- binding site
- carbon nuclear magnetic resonance
- carboxy terminal sequence
- enzyme binding
- enzyme conformation
- hydrogen bond
- microenvironment
- nitrogen nuclear magnetic resonance
- nonhuman
- pH measurement
- pKa
- priority journal
- protein degradation
- protein family
- protein interaction
- proton nuclear magnetic resonance
- proton transport
- titrimetry