Glucuronoxylan Xylanohydrolase A UNIQUE XYLANASE WITH THE REQUIREMENT FOR APPENDANT GLUCURONOSYL UNITS*

A new category of @-(1+4)-xylan xylanohydrolases that exhibit a specific capacity to hydrolyze glucuronoxylans was characterized using heteroxylans prepared from Vigna (Vigna angularis Ohwi et Ohashi cv. Takara) and maize (Zea mays L.) cell walls together with appropriate derivatives as substrates. Glucuron-opyranosyl moieties, as side chains, were prerequisite for enzyme-mediated hydrolysis of the &( l+4)-xylo-syl linkages. The enzyme degraded glucuronoxylans derived from Vigna cell walls to yield a major oligomeric species where Xyl represents xylose and GlcA represents glucuronic acid. The enzyme also degraded glucuronoarabinoxylans derived from maize cell walls to yield a major oligomeric species containing a single glucuronosyl side chain and a single unsubstituted j31+4Xyl pendant terminal. These results indicate that this xylanohydrolase recognizes glucuronosyl moieties in-serted as monomeric side chains along the xylan backbone and mediates the hydrolysis of the &(1+4)-xylo-syl

t where Xyl represents xylose and GlcA represents glucuronic acid. The enzyme also degraded glucuronoarabinoxylans derived from maize cell walls to yield a major oligomeric species containing a single glucuronosyl side chain and a single unsubstituted j31+4Xyl pendant terminal. These results indicate that this xylanohydrolase recognizes glucuronosyl moieties inserted as monomeric side chains along the xylan backbone and mediates the hydrolysis of the &(1+4)-xylosyl linkage of the adjacent unsubstituted xylosyl residue in heteroxylans. This enzyme is the first xylanohydrolase identified that recognizes distinctly different sugars constituting side chains. We propose to designate this new enzyme as a glucuronoxylan xylanohydrolase to be abbreviated as glucuronoxylanase.
Use of this unique enzyme demonstrated the presence of repeating units in heteroxylans in cell walls of higher plants.
Xylans are major components of the matrix of most higher plant cell walls and are characterized as @-(1+4)-xylopyranosyls (1). These xylans are often highly substituted with 2 To whom correspondence should be addressed. glucuronic acid (GlcA),' Ara, and feruloyl residues to form complex heteroxylans (2)(3)(4)(5). As a component of primary cell walls of plants (6,7), the polymer is considered to serve an important role in controlling wall functions (7,8) and is thought to participate in processes responsible for growth and differentiation (7)(8)(9)(10).
A Bacillus subtilis enzyme preparation provided a source for the purification of unique xylanohydrolases (23). These enzymes showed the high specificity for degradation of feruloylated glucuronoarabinoxylans ("feraxans") and were tentatively designated feraxanases (23). Because of the capacity of the enzyme to fragment the xylan, it was employed for structural analysis of feraxan in maize cells walls (4,7). Although it was suggested that the enzymic action was dependent on certain structural features along the xylan main chain, the nature of the side chain required for recognition and the mode of action of the enzyme was unclear (23). We have now characterized the substrate specificity for this enzyme and identified the recognition site in selected heteroxylans. The enzyme recognizes GlcA side chains along the xylan main chain and mediates the hydrolysis of the @-(I-4)-xylosyl linkages of the adjacent unsubstituted xylosyl residues.
The abbreviations used are: GlcA, glucuronic acid; Xyl, xylose; Ara, arabinose; GC, gas chromatography; GPC, gel permeation chromatography; HPLC, high performance liquid chromatography; RI, refractive index; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; MX, maize xylan; VX, Vigna xylan; MXf and VXf, the major fragments obtained following B. subtilis glucuronoxylan xylanohydrolase digestion of MX and VX, respectively; -CR, poly-or oligosaccharides in which carboxyl groups in uronosyl residues and the hemiacetal at the reducing end is reduced; -A, polysaccharides in which arabinosyl residues are partially hydrolyzed -OH, poly-or oligosaccharides whose hemiacetals at the reducing ends are reduced. Profiles of the feraxanase digestion products released from maize xylan as a function of reaction time. One mg of MX was incubated with 100 ng of feraxanase for the periods indicated, and the products were analyzed by Superose 12 (GPC system A). Chromatographic conditions are described under "Experimental Procedures." RID, RI detector; RT, retention time.

RESULTS AND DISCUSSION
Substrate Specificity-Feraxanase degraded MX and VX to yield products resolved by Superose 12 (system A) (Fig. 6). An enzymic degradation pattern as a function of time reveals the progression of fragment release (Fig. 7). The appearance of intermediate sized fragments at an early stage of the reaction is consistent with an endo type action pattern. An in  6). However, xylan derivatives whose GlcA residues had been converted to Glc residues were no longer substrates for the enzyme (MX-CR, MX-A-CR, and VX-CR) (Fig. 6). Modification of other side chain constituents in xylans, ,i.e. the deletion of most (83%) of the Ara side chain or reduction of the hemiacetal at the reducing end residue, did not influence the enzyme's ability to act on substrate derivatives. The results demonstrate that GlcA side chains are essential for feraxanase-mediated hydrolysis. Recognition Site-Exhaustive digestion of VX (30 mg) with feraxanase yielded a major oligomer fraction (14.2 mg) designated as VXf fraction (Fig. 2). The GlcA residue and hemiacetal of the reducing end of VXf was reduced to obtain VXf-CR (Table 11, Fig. 3). Hydrolysis of VXf-CR with an endop(lA)-xylanase gave two fragments, VXf-CR-x1 and -x2. These fragments were identified as Xyl/314(Glcla2)xylitol and Xyl/31+4Xyl/31+4Xyl, respectively, from interpretation of fragments prepared after treatment with purified /3-xylosidase (Fig. 4), methylation analyses (Table 11), and 13C NMR spectroscopy. Based on these results, VXf was identified as Xyl~l+4Xyl~l+4Xyl/31+4Xyl/31+4(GlcAa1+2)Xyl/3l+ 4Xyl. On the other hand, digestion of MX with feraxanase gave a major oligosaccharide fraction designated as MXf (1.6 kDa), which contained both a single GlcA residue and an unsubstituted pendent (1+4)-Xyl residue (Table 111, Fig. 5). Thus, common structural features of VXf and MXf are a single GlcA side chain and an unsubstituted (1+4)-Xyl reducing end.
This line of evidence supports the idea that the enzyme recognizes a GlcA moiety on the xylan main chain and hydrolyzes a p-(1+4)-Xyl linkage in the vicinity of the GlcA side chain. Two possible recognition sites of the enzyme are shown in Fig. 8. One is that the enzyme recognizes the GlcA moiety at site C in Fig. 8 and hydrolyzes the xylosyl linkage at site A (reducing end side). The alternative idea is that the enzyme recognizes at site C and hydrolyzes the linkage at site B (nonreducing end side). The K,,, value for VX-OH (5.0 mg/ ml) was similar to that for MX-OH (6.9 mg/ml). In MX-OH, Xyl residues located between site B and site C were highly substituted with Ara side chains, whereas the Xyl residue between sites A and C was not substituted. On the other hand, VX-OH was not substituted with Ara. These data indicate that the Ara side chains located between sites B and C do not affect the affinity of the enzyme to the polysaccharide. Based on these results, we conclude that the enzyme recognizes GlcA residue (site C) and splits the p-(1+4)-Xyl linkage (site A) of the adjacent unsubstituted Xyl residue located at the reducing end side of the xylan.
Thus, appendages to the basic structure are not a prerequisite for catalysis by these xylanases.

Presence of Repeating Structural Units in Heteroxylans-
This study demonstrates for the first time the presence of repeating structural units as major components in xylans derived both from maize and Vigna cell walls; GlcA side chains regularly distributed along P-(l+4)-xylans serve as markers. This finding evokes a consideration of the presence of a regulatory mechanism for processing heteroxylan during synthesis and degradation in plant tissues. The evidence clearly vindicates the usefulness of this enzymic approach as a powerful new tool for specific fragmentation of heteroxylans. The enzymic fragmentation of cell wall macromolecules offers the opportunity for a unique approach for exploring the structure of a major component of plant cell walls. v