Abstract
Glucuronoxylanases are endo-xylanases and members of the glycoside hydrolase family 30 subfamilies 7 (GH30-7) and 8 (GH30-8). Unlike for the well-studied GH30-8 enzymes, the structural and functional characteristics of GH30-7 enzymes remain poorly understood. Here, we report the catalytic properties and three-dimensional structure of GH30-7 xylanase B (Xyn30B) identified from the cellulolytic fungus Talaromyces cellulolyticus Xyn30B efficiently degraded glucuronoxylan to acidic xylooligosaccharides (XOSs), including an α-1,2-linked 4-O-methyl-d-glucuronosyl substituent (MeGlcA). Rapid analysis with negative-mode electrospray-ionization multistage MS (ESI(-)-MS n ) revealed that the structures of the acidic XOS products are the same as those of the hydrolysates (MeGlcA2Xyl n , n > 2) obtained with typical glucuronoxylanases. Acidic XOS products were further degraded by Xyn30B, releasing first xylobiose and then xylotetraose and xylohexaose as transglycosylation products. This hydrolase reaction was unique to Xyn30B, and the substrate was cleaved at the xylobiose unit from its nonreducing end, indicating that Xyn30B is a bifunctional enzyme possessing both endo-glucuronoxylanase and exo-xylobiohydrolase activities. The crystal structure of Xyn30B was determined as the first structure of a GH30-7 xylanase at 2.25 Å resolution, revealing that Xyn30B is composed of a pseudo-(α/β)8-catalytic domain, lacking an α6 helix, and a small β-rich domain. This structure and site-directed mutagenesis clarified that Arg46, conserved in GH30-7 glucuronoxylanases, is a critical residue for MeGlcA appendage-dependent xylan degradation. The structural comparison between Xyn30B and the GH30-8 enzymes suggests that Asn93 in the β2-α2 loop is involved in xylobiohydrolase activity. In summary, our findings indicate that Xyn30B is a bifunctional endo- and exo-xylanase.
Highlights
Glucuronoxylanases are endo-xylanases and members of the glycoside hydrolase family 30 subfamilies 7 (GH30-7) and 8 (GH30-8)
This paper described the characterization and structural determination of the novel GH30-7 xylanase, Xyn30B, from T. cellulolyticus
Xyn30B displayed glucuronoxylan-specific endo-xylanase activity that has been reported in bacterial GH30 subfamilies 8 (GH30-8) glucuronoxylanases and T. reesei GH30-7 XYN VI
Summary
The Xyn30B gene is composed of 1,425 bp without introns in the T. cellulolyticus genome and encodes a protein consisting of 474 amino acid residues. Xyn30B has a relatively high amino acid sequence identity with fungal GH30-7 enzymes, such as XYN IV (38.2%) and XYN VI (42.2%) from T. reesei and XYLD (53.4%) from Bispora sp. As with XYN IV and XYN VI, an Arg residue responsible for recognition of the MeGlcA in GH30-8 enzymes is not conserved in Xyn30B (Fig. 1, red box). The Xyn30B amino acid sequence includes a signal sequence (residues 1–22) as predicted by the SignalP server [23]. SDS-PAGE analysis of the purified enzyme showed a molecular mass slightly larger than 49,403 Da, which has been estimated from the primary structure excluding the N-terminal signal peptide (Fig. 2). The glycosylation sites in Xyn30B were assigned by X-ray crystallography, as described below
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