Structural Dynamics and Catalytic Properties of a Multimodular Xanthanase

Abstract

The precise catalytic strategies used for the breakdown of the complex bacterial polysaccharide xanthan, an increasingly frequent component of processed human foodstuffs, have remained a mystery. Here we present the characterization of an endo-xanthanase from Paenibacillus sp. 62047. We show that it is a CAZy family 9 glycoside hydrolase (GH9) responsible for the hydrolysis of the xanthan backbone, capable of generating tetrameric xanthan oligosaccharides from polysaccharide lyase family 8 (PL8) xanthan lyase-treated xanthan. 3-D structure determination reveals a complex multi-modular enzyme in which a catalytic (α/α)6 barrel is flanked by an N-terminal "immunoglobulin-like" (Ig-like) domain (frequently found in GH9 enzymes) and by four additional C-terminal all β-sheet domains which have very few homologs in sequence databases and, at least, one of which functions as a new xanthan-binding domain, now termed CBM84. The solution phase conformation and dynamics of the enzyme in the native calcium-bound state and in the absence of calcium were probed experimentally by hydrogen/deuterium exchange mass spectrometry. Measured conformational dynamics were used to guide the protein engineering of enzyme variants with increased stability in the absence of calcium; a property of interest for the potential use of the enzyme in cleaning detergents. The ability of hydrogen/deuterium exchange mass spectrometry to pinpoint dynamic regions of a protein under stress (e.g. removal of calcium ions) makes this technology a strong tool for improving protein catalyst properties by informed engineering.

The Food Additive Xanthan Gum Drives Adaptation of the Human Gut Microbiota

“…Surprisingly, the NMR analysis suggested that Ru GH5a cleaves XG at the reducing end of the non-branching backbone glucosyl residue ( Figure 3c, Extended Data 7 ). This contrasts with the product of other known xanthanases (such as the GH9 from Paenibacillus nanensis 35 or the β-D-glucanase in Bacillus sp. strain GL1 15 ), which hydrolyze xanthan at the reducing end of the branching glucose, demonstrating a hitherto unknown enzymatic mechanism for the degradation of XG.…”

“…strain GL1 15 ), which hydrolyze xanthan at the reducing end of the branching glucose, demonstrating a hitherto unknown enzymatic mechanism for the degradation of XG. While Ru GH5a displayed little activity on other polysaccharides ( Extended Data 6 ), it was able to hydrolyze both native and lyase-treated XG with comparable specificity, once more in contrast to most previously known xanthanases, which show ≥600-fold preference for the lyase-treated substrate 35 ( Figure 3d ). One exception is the xanthanase from Microbacterium sp.…”

Structure and Dynamics of a Promiscuous Xanthan Lyase from Paenibacillus nanensis and the Design of Variants with Increased Stability and Activity

“…This endo-xanthanase is able to attack the backbone of XL-treated xanthan and possesses a CD belonging to glycoside hydrolase family 9 along with four additional domains constituted largely of b sheets. The latter of these corresponds to the module identified here, and has now been shown to bind xanthan, becoming the founding member of CBM family 84 (Moroz et al, 2018). Structurally related to CBM6, CBM35, and CBM36, the structure of PXL CBM84 is shown in Figure 2.…”

“…As seen in the Results section, CBM84 is essential for full activity by PXL. Interestingly, truncation of the corresponding module did not impair the activity of the endo-xanthanase in Moroz et al (2018). Since initial sequence analysis indicated a relationship of CBM84 and X to CBM35, which are related to CBM6 and CBM36, pairwise structural similarity comparisons were carried out for both modules against all representatives of CBM6, CBM35, and CBM36.…”

“…Structurally related to CBM6, CBM35, and CBM36, the structure of PXL CBM84 is shown in Figure 2. Notably, the structure of the corresponding module in the endo-xanthanase could not be fully structurally characterized due to disorder (Moroz et al, 2018). The second module (referred to here as X) can be aligned with CBM84 with C a root-mean-square deviation (RMSD) of 2.51 A ˚over 119 out of 136 residues (Data S1) and the two modules are thus structurally related.…”