Structure features of GH10 xylanase from <italic>Caldicellulosiruptor bescii</italic>: implication for its thermophilic adaption and substrate binding preference

Abstract

Caldicellulosiruptor bescii is the most thermophilic cellulolytic species of organisms known to date. In our previous study, GH10 xylanase CbXyn10B from C. bescii displayed outstanding hydrolytic activity toward various xylans at high temperatures. To understand the structural basis for this protein's catalysis and thermostability, we solved the crystal structures of CbXyn10B and its complexes with xylooligosaccharides. These structural models were used to guide comparison with its mesophilic counterpart PbXyn10B. A distinctive structural feature is that thermophilic CbXyn10B presents a relatively stable interaction between the extended loops L7 and L8 in the catalytic cleft by an extensive hydrogen bonding network, which is mediated by Lys, Arg and three well-ordered water molecules. Moreover, a unique aromatic cluster consisting of Try, Phe, Phe, and Phe may enhance the interaction between the N- and C- terminus. Targeted mutagenesis demonstrated that these interactions substantially contribute to enzyme stabilization, as indicated by a considerable decrease in the melting temperature (T) of CbXyn10B by substituting critical residues with Ala. Therefore, it was shown that not only the aromatic interaction connecting protein termini but also the extensive hydrogen bonding network formed between surface loops could restrict the local structural flexibility and contribute significantly to the overall stability of enzymes. Furthermore, the xylooligosaccharides were found to tightly bind to the glycone subsites of xylanase, indicating higher affinities at these subsites and reflecting its substrate binding preference. Our results suggest that CbXyn10B is stabilized with distinct rigidity at the catalytic cleft as well as the terminal regions, which provides insights into the evolutionary strategy for accommodating the functional needs of GH10 enzymes to high temperature.

A detailed overview of xylanases: an emerging biomolecule for current and future prospective

“…It usually consists of high-molecular weight xylanase with low isoelectric points and displays an (α/β) 8 -barrel fold. This structure mimics the shape of a 'Salad Bowl' , because of an enlarged loop architecture, one face of the molecule is having ~ 45 Å large radius and the other face is having ~ 30 Å radius because of simple (α/β) turns (Zhang et al 2016a(Zhang et al , 2016b. However, these two categories are relatively the same because along with sharing similar fold also shares some common residues and has similar catalytic mechanisms.…”

“…Hence, for complete and efficient hydrolysis of xylan into its constituent sugars requires synergistic action of various enzymes with specifically targeting appropriate bonds of xylan. The multifunction xylanolytic system exists in bacteria (Zhang et al 2016a(Zhang et al , 2016b, fungi (Driss et al 2011;) and actinomycetes (Hunt et al 2016) where xylan backbone is randomly cleaved by the action of endo-1, 4-β-d-xylanases; xylose polymer is broken down to its monomeric form by action of β-dxylosidases. Acetyl and phenolic side branches were removed by the action of α-glucuronidase and acetylxylan esterase.…”

Characterization of efficient xylanases from industrial-scale pulp and paper wastewater treatment microbiota

“…Xylanases of the GH10 family, which normally have high molecular weight and a (β/α) 8 8-barrel folding structure, hydrolyze xylan through a reservation-type catalytic mechanism (Kim et al 2018;Zhang et al 2016). Crystal structure and dynamics analysis have revealed that these xylanases have four to five active binding sites, and often contain carbohydrate-binding modules (CBM) connected to their catalytic structural domain (Jia and Han 2019; Wu et al 2020).…”

Structure-function relationship of bifunctional XynA

“…The xylanase GH10A is capable of degrading wheat arabinoxylan (WAX) and tamarind xyloglucan and it is difficult to detect the binding ability (Fredriksen et al, 2019). The bifunctional enzymes with xylanase/cellulase activity have been reported such as CbXyn10C (5OFJ) (Chu et al, 2017), SlXyn10A (1E0X) (Ducros et al, 2000a), CbXyn10B (4L4O) (Zhang et al, 2016) and CfCex (Liu, Zhang, & Xu, 2012). CbXyn10C, Xyl10A, and CbXyn10B all have the classic (α/β) 8 TIM-barrel fold structure, but there is no article revealing the "switch" of the GH10 family of xylanases from single function to dual function.…”