Mode of action of GH30-7 reducing-end xylose-releasing exo-xylanase A (Xyn30A) from the filamentous fungus Talaromyces cellulolyticus

In this study, we characterized the mode of action of reducing-end xylose-releasing exo-xylanase (Rex), which belongs to the glycoside hydrolase family (GH) 30-7. GH30-7 Rex, isolated from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), exists as a dimer. The purified Xyn30A released xylose from the linear xylooligosaccharides (XOSs) 3 to 6 xylose units in length with similar kinetic constants. Hydrolysis of branched, borohydride-reduced, and p -nitrophenyl XOSs clarified that Xyn30A possesses a Rex activity. 1 H NMR analysis of xylotriose hydrolysate indicated that Xyn30A degraded XOSs via retaining mechanism and without recognizing an anomeric structure at the reducing end. Hydrolysis of xylan by Xyn30A revealed that the enzyme continuously liberated both xylose and two types of acidic XOSs: 2 2 -(4- O -methyl-α-d-glucuronyl)-xylotriose (MeGlcA 2 Xyl 3 ) and 2 2 -(MeGlcA)-xylobiose (MeGlcA 2 Xyl 2 ). These acidic products were also detected during hydrolysis using a mixture of MeGlcA 2 Xyl n (n = 2–14) as substrate. This indicates that Xyn30A can release MeGlcA 2 Xyl n (n = 2 and 3) in an exo-manner. Comparison of subsites in Xyn30A and GH30-7 glucuronoxylanase using homology modeling suggested that the binding of reducing-end residue at subsite +2 was partially prevented by a Gln residue conserved in GH30-7 Rex; additionally, the Arg residue at subsite -2b, conserved in glucuronoxylanase, was not found in Xyn30A. Our results propose that GH30-7 Rex plays a complementary role in hydrolysis of xylan by fungal cellulolytic systems. Importance Endo- and exo-type xylanases depolymerize xylan and play crucial roles in the assimilation of xylan in bacteria and fungi. Exo-xylanases release xylose from the reducing or non-reducing end of xylooligosaccharides; this is generated by the activity of endo-xylanases. β-xylosidase, which hydrolyzes xylose residues on the non-reducing end of a substrate, is well-studied. However, the function of reducing-end xylose-releasing exo-xylanases (Rex), especially in fungal cellulolytic systems, remains unclear. This study revealed the mode of xylan hydrolysis by Rex from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), which belongs to the glycoside hydrolase family 30-7 (GH30-7). A conserved residue related to Rex activity is found in the substrate-binding site of Xyn30A. These findings will enhance our understanding of the function of GH30-7 Rex in the cooperative hydrolysis of xylan by fungal enzymes.