Structural analysis of the reducing-end xylose-releasing exo-oligoxylanase Rex8A from Paenibacillus barcinonensis BP-23 deciphers its molecular specificity.

Reducing-end xylose-releasing exo-oligoxylanases (Rex) are GH8 enzymes that depolymerize xylooligosaccharides complementing xylan degradation by endoxylanases in an exo manner. We have studied Paenibacillus barcinonensis Rex8A and showed the release of xylose from xylooligomers decorated with methylglucuronic acid (UXOS) or with arabinose (AXOS). This gives the enzyme a distinctive trait among known Rex, which show activity only on linear xylooligosaccharides. The structure of the enzyme has been solved by X-ray crystallography showing a (α/α)6 folding common to GH8 enzymes. Analysis of inactived Rex8A-E70A complexed with xylotetraose revealed the existence of at least four binding subsites in Rex8A, with the oligosaccharide occupying subsites -3 to +1. The enzyme shows an extended Leu320-His321-Pro322 loop, common to other Rex, which blocks the binding of longer substrates to positive subsites further than +1 and seems responsible for the lack or diminished activity of Rex enzymes on xylan. Mutants with smaller residues in this loop failed to increase Rex8A activity on the polymer. Analysis of the complexes with AXOS showed the accommodation of arabinose at subsite -2, which cannot be allocated at subsite -1. Arabinose substitutions at the xylose O2 or O3 are accommodated by hydrophobic interaction and seem tolerated rather than recognized by Rex8A. A strained binding of the branch is facilitated by the lack of direct polar interactions of the xylose occupying this subsite, its water-mediated links allowing some conformational flexibility of the sugar. The plasticity of Rex8A is a notable property of the enzyme for its application in xylan deconstruction and upgrading. DATABASE: Structural data are available in PDB database under the accession numbers 6SRD (native form), 6TPP (E70A mutant in complex with EDO), 6TOW (E70A in complex with Xyl4), 6SUD (L320A mutant in complex with xylose), 6SHY (L320A/H321S double mutant in complex with EDO), 6TO0 (E70A in complex with AX3), and 6TRH (E70A in complex with AX4).

Jiménez-Ortega E ，Valenzuela S ，Ramírez-Escudero M ，Pastor FJ ，Sanz-Aparicio J ... -  《-》

The Glycoside Hydrolase Family 8 Reducing-End Xylose-Releasing Exo-oligoxylanase Rex8A from Paenibacillus barcinonensis BP-23 Is Active on Branched Xylooligosaccharides.

A GH8 family enzyme involved in xylan depolymerization has been characterized. The enzyme, Rex8A, is a reducing-end xylose-releasing exo-oligoxylanase (Rex) that efficiently hydrolyzes xylooligosaccharides and shows minor activity on polymeric xylan. Rex8A hydrolyzes xylooligomers of 3 to 6 xylose units to xylose and xylobiose in long-term incubations. Kinetic constants of Rex8A were determined on xylotriose, showing a Km of 1.64 ± 0.03 mM and a kcat value of 118.8 s(-1) Besides linear xylooligosaccharides, the enzyme hydrolyzed decorated xylooligomers. The catalytic activity on branched xylooligosaccharides, i.e., the release of xylose from the reducing end, is a newly described trait of xylose-releasing exo-oligoxylanases, as the exo-activity on these substrates has not been reported for the few of these enzymes characterized to date. Modeling of the three-dimensional (3D) structure of Rex8A shows an (α/α)6 barrel fold where the loops connecting the α-helices contour the active site. These loops, which show high sequence diversity among GH8 enzymes, shape a catalytic cleft with a -2 subsite that can accommodate methyl-glucuronic acid decorations. The hydrolytic ability of Rex8A on branched oligomers can be crucial for the complete depolymerization of highly substituted xylans, which is indispensable to accomplish biomass deconstruction and to generate efficient catalysts. A GH8 family enzyme involved in xylan depolymerization has been characterized. The Rex8A enzyme from Paenibacillus barcinonensis is involved in depolymerization of glucuronoxylan, a major component of the lignocellulosic substrates. The study shows that Rex8A is a reducing-end xylose-releasing exo-oligoxylanase that efficiently hydrolyzes xylose from neutral and acidic xylooligosaccharides generated by the action of other xylanases also secreted by the strain. The activity of a Rex enzyme on branched xylooligosaccharides has not been described to date. This report provides original and useful information on the properties of a new example of the rarely studied Rex enzymes. Depolymerization of highly substituted xylans is crucial for biomass valorization as a platform for generation of biofuels, chemicals, and solvents.

Valenzuela SV ，Lopez S ，Biely P ，Sanz-Aparicio J ，Pastor FI ... -  《-》

A novel reducing-end xylose-releasing exo-oligoxylanase (PphRex8A) from Paenibacillus physcomitrellae XB.

In order to better understand the function of a putative GH8 xylanase gene in the xylan-degrading bacterium Paenibacillus physcomitrellae XB, a novel reducing-end xylose-releasing exo-oligoxylanase (Rex) PphRex8A of GH8 family was characterized. Phylogenetic analysis showed that it was clustered tightly with other published GH8 Rexs and exhibited the highest amino acid sequence identity (77.4 %) with the Rex of PbRex8 from P. barengoltzii G22. The three-dimensional (3D) structure of PphRex8A was also built based on the template of PbRex8 (5YXT) and three conserved catalytic active sites (Glu71, Asp263, and Asp129) were predicted and further confirmed by the enzymatic inactivity of their mutants (E71A, D129A, and D263A). The hydrolysis assay of PphRex8A showed that it could hydrolyze xylo-oligosaccharides (XOSs) with a degree of polymerization (DP) ≥ 3, such as xylotriose (X3) through xylohexaose (X6), and some natural XOSs, such as corncob xylan (CCX) and oat spelt xylan (OSX) to release xylose. However, it could not hydrolyze p-nitrophenyl-β-D-xylopyranoside (pNPX), which suggested that it mainly released xylose from the reducing-end of XOSs and belonged to a Rex. In addition, PphRex8A also could deconstruct xylans with high DP, such as wheat arabinoxylan (AX) and beech wood xylan (BWX) to produce XOSs with DP3-6. Moreover, PphRex8A had synergistic effects with other xylanolytic enzymes of P. physcomitrellae XB, such as with PphXyn10 or PphXyn11 at a ratio of 1:3, or with PphXyl43B as a ratio of 3:1, significantly increasing the amounts of reducing sugars toward different xylan substrates. Thus, PphRex8A could be an exo-xylanase toward XOSs and could improve the deconstruction capability of high DP xylans, thereby complementing other xylanolytic enzymes to contribute to xylan degradation and improve the efficiency of converting hemicellulose biomass into energy by P. physcomitrellae XB.

Wang L ，Zhang XJ ，Li YH 《-》

Structural basis for the specificity of the reducing end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125.

Fushinobu S ，Hidaka M ，Honda Y ，Wakagi T ，Shoun H ，Kitaoka M ... -  《JOURNAL OF BIOLOGICAL CHEMISTRY》

Mode of Action of GH30-7 Reducing-End Xylose-Releasing Exoxylanase A (Xyn30A) from the Filamentous Fungus Talaromyces cellulolyticus.

In this study, we characterized the mode of action of reducing-end xylose-releasing exoxylanase (Rex), which belongs to the glycoside hydrolase family 30-7 (GH30-7). GH30-7 Rex, isolated from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), exists as a dimer. The purified Xyn30A released xylose from 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. 1H nuclear magnetic resonance (1H NMR) analysis of xylotriose hydrolysate indicated that Xyn30A degraded XOSs via a 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: 22-(4-O-methyl-α-d-glucuronyl)-xylotriose (MeGlcA2Xyl3) and 22-(MeGlcA)-xylobiose (MeGlcA2Xyl2). These acidic products were also detected during hydrolysis using a mixture of MeGlcA2Xyl n (n = 2 to 14) as the substrate. This indicates that Xyn30A can release MeGlcA2Xyl 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 the 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, which is conserved in glucuronoxylanase, was not found in Xyn30A. Our results lead us to 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. Exoxylanases release xylose from the reducing or nonreducing ends of xylooligosaccharides; this is generated by the activity of endoxylanases. β-Xylosidase, which hydrolyzes xylose residues on the nonreducing end of a substrate, is well studied. However, the function of reducing-end xylose-releasing exoxylanases (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.

Nakamichi Y ，Fouquet T ，Ito S ，Matsushika A ，Inoue H ... -  《-》