Bifunctional Family 3 Glycoside Hydrolases from Barley with α-l-Arabinofuranosidase and β-d-Xylosidase Activity
2003; Elsevier BV; Volume: 278; Issue: 7 Linguagem: Inglês
10.1074/jbc.m210627200
ISSN1083-351X
AutoresRobert C. Lee, Mária Hrmová, Rachel A. Burton, Jelle Lahnstein, Geoffrey B. Fincher,
Tópico(s)Microbial Metabolites in Food Biotechnology
ResumoAn α-l-arabinofuranosidase and a β-d-xylosidase, designated ARA-I and XYL, respectively, have been purified about 1,000-fold from extracts of 5-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, ion exchange chromatography, chromatofocusing, and size-exclusion chromatography. The ARA-I has an apparent molecular mass of 67 kDa and an isoelectric point of 5.5, and its catalytic efficiency during hydrolysis of 4′-nitrophenyl α-l-arabinofuranoside is only slightly higher than during hydrolysis of 4′-nitrophenyl β-d-xyloside. Thus, the enzyme is actually a bifunctional α-l-arabinofuranosidase/β-d-xylosidase. In contrast, the XYL enzyme, which also has an apparent molecular mass of 67 kDa and an isoelectric point of 6.7, preferentially hydrolyzes 4′-nitrophenyl β-d-xyloside, with a catalytic efficiency ∼30-fold higher than with 4′-nitrophenyl α-l-arabinofuranoside. The enzymes hydrolyze wheat flour arabinoxylan slowly but rapidly hydrolyze oligosaccharide products released from this polysaccharide by (1 → 4)-β-d-xylan endohydrolase. Both enzymes hydrolyze (1 → 4)-β-d-xylopentaose, and ARA-I can also degrade (1 → 5)-α-l-arabinofuranohexaose. ARA-I and XYL cDNAs encode mature proteins of 748 amino acid residues which have calculated molecular masses of 79.2 and 80.5 kDa, respectively. Both are family 3 glycoside hydrolases. The discrepancies between the apparent molecular masses obtained for the purified enzymes and those predicted from the cDNAs are attributable to COOH-terminal processing, through which about 130 amino acid residues are removed from the primary translation product. The genes encoding the ARA-I and XYL have been mapped to chromosomes 2H and 6H, respectively. ARA-I transcripts are most abundant in young roots, young leaves, and developing grain, whereas XYL mRNA is detected in most barley tissues. An α-l-arabinofuranosidase and a β-d-xylosidase, designated ARA-I and XYL, respectively, have been purified about 1,000-fold from extracts of 5-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, ion exchange chromatography, chromatofocusing, and size-exclusion chromatography. The ARA-I has an apparent molecular mass of 67 kDa and an isoelectric point of 5.5, and its catalytic efficiency during hydrolysis of 4′-nitrophenyl α-l-arabinofuranoside is only slightly higher than during hydrolysis of 4′-nitrophenyl β-d-xyloside. Thus, the enzyme is actually a bifunctional α-l-arabinofuranosidase/β-d-xylosidase. In contrast, the XYL enzyme, which also has an apparent molecular mass of 67 kDa and an isoelectric point of 6.7, preferentially hydrolyzes 4′-nitrophenyl β-d-xyloside, with a catalytic efficiency ∼30-fold higher than with 4′-nitrophenyl α-l-arabinofuranoside. The enzymes hydrolyze wheat flour arabinoxylan slowly but rapidly hydrolyze oligosaccharide products released from this polysaccharide by (1 → 4)-β-d-xylan endohydrolase. Both enzymes hydrolyze (1 → 4)-β-d-xylopentaose, and ARA-I can also degrade (1 → 5)-α-l-arabinofuranohexaose. ARA-I and XYL cDNAs encode mature proteins of 748 amino acid residues which have calculated molecular masses of 79.2 and 80.5 kDa, respectively. Both are family 3 glycoside hydrolases. The discrepancies between the apparent molecular masses obtained for the purified enzymes and those predicted from the cDNAs are attributable to COOH-terminal processing, through which about 130 amino acid residues are removed from the primary translation product. The genes encoding the ARA-I and XYL have been mapped to chromosomes 2H and 6H, respectively. ARA-I transcripts are most abundant in young roots, young leaves, and developing grain, whereas XYL mRNA is detected in most barley tissues. Heteroxylans are major constituents of cell walls in the Poaceae, which include many commercially important cereals and pasture grasses. In the endosperm of barley grains and in elongating coleoptiles, these polysaccharides may comprise 20–70% by weight of the walls (1Fincher G.B. Shewry P.R. Barley: Genetics, Molecular Biology and Bio/Technology. CAB Publishing, Wallingford, U. K.1992: 413-437Google Scholar) and consist of a backbone of (1 → 4)-β-linkedd-xylopyranosyl residues substituted predominantly with α-l-arabinofuranosyl residues. The α-l-arabinofuranosyl residues can be linked to O-3, O-2, or both O-3 and O-2 of xylanopyranosyl residues of the (1 → 4)-β-d-xylan backbone, and other substituents or short side chains are also detected in low abundance (2Viëtor R.J. Kormelink F.J.M. Angelino S.A.G.F. Voragen A.G.J. Carbohydr. Polym. 1994; 24: 113-118Crossref Scopus (35) Google Scholar, 3Carpita N.C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 445-476Crossref PubMed Scopus (681) Google Scholar). The α-l-arabinofuranosyl residues can be esterified with hydroxycinnamic acids, in particular ferulic acid, which may form cross-bridges between adjacent arabinoxylan chains, or with lignin, by oxidative dimerization (4Iiyama K. Lam T.B.T. Stone B.A. Plant Physiol. 1993; 104: 315-320Crossref Scopus (639) Google Scholar).As observed with many wall components in higher plants, the arabinoxylans of primary cell walls can be restructured during normal growth and development. For example, newly synthesized arabinoxylans in maize coleoptiles are deposited in the walls in a highly substituted form, but arabinofuranosyl residues are removed later, and this leads to significant changes in the physicochemical properties of the polysaccharides and hence in the walls themselves (5Andrewartha K. Phillips D.R. Stone B.A. Carbohydr. Res. 1979; 77: 191-204Crossref Scopus (207) Google Scholar, 6Gibeaut D.M. Carpita N.C. Plant Physiol. 1991; 97: 551-561Crossref PubMed Scopus (187) Google Scholar). Removal of α-l-arabinofuranosyl residues is also observed when wall arabinoxylans are degraded (7Wilkie K.C.B. Adv. Carbohydr. Chem. Biochem. 1979; 36: 215-264Crossref Scopus (330) Google Scholar, 8Heyn A.N.J. Plant Sci. 1986; 45: 77-82Crossref Scopus (8) Google Scholar). The presence of α-l-arabinofuranosidases in germinated barley grain or in isolated aleurone layers has been taken as evidence that these enzymes perform this function during the mobilization of the starchy endosperm after cereal grain germination (9Taiz L. Honigman W.A. Plant Physiol. 1976; 58: 380-386Crossref PubMed Google Scholar, 10Banik M. Li C.D. Langridge P. Fincher G.B. Mol. Gen. Genet. 1997; 253: 599-608Crossref PubMed Scopus (54) Google Scholar), although this activity has also been attributed to a separate group of enzymes, known as arabinoxylan α-l-arabinofuranohydrolases (11Ferré H. Broberg A. Duus J.Ø. Thomsen K.K. Eur. J. Biochem. 2000; 267: 6633-6641Crossref PubMed Scopus (73) Google Scholar, 12Lee R.C. Burton R.A. Hrmova M. Fincher G.B. Biochem. J. 2001; 356: 181-189Crossref PubMed Scopus (84) Google Scholar).Most α-l-arabinofuranosidases are so designated because they can hydrolyze the synthetic aryl glycoside 4′-nitrophenyl α-l-arabinofuranoside (4NPA), 1The abbreviations used are: 4NPA, 4′-nitrophenyl α-l-arabinofuranoside; ARA-I, α-l-arabinofuranosidase; HCA, hydrophobic cluster analysis; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; 4NPX, 4′-nitrophenyl β-d-xylopyranoside; RACE, rapid amplification of cDNA ends; RFLP, restriction fragment length polymorphism; XYL, β-d-xylosidase 1The abbreviations used are: 4NPA, 4′-nitrophenyl α-l-arabinofuranoside; ARA-I, α-l-arabinofuranosidase; HCA, hydrophobic cluster analysis; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; 4NPX, 4′-nitrophenyl β-d-xylopyranoside; RACE, rapid amplification of cDNA ends; RFLP, restriction fragment length polymorphism; XYL, β-d-xylosidase and although they are presumed to be responsible for changes in arabinoxylans during wall modification or degradation, this class of enzymes may be subdivided into several quite distinct groups. Thus, α-l-arabinofuranosidases have been classified in glycoside hydrolase families 3, 43, 51, 54, and 62 (13; afmb.cnrs-mrs.fr), and members of each family exhibit characteristic substrate specificities, action patterns, and reaction mechanisms (14Pitson S.M. Voragen A.G.J. Beldman G. FEBS Lett. 1996; 398: 7-11Crossref PubMed Scopus (52) Google Scholar, 15McKie V.A. Black G.W. Millward-Sadler S.J. Hazlewood G.P. Laurie J.I. Gilbert H.J. Biochem. J. 1997; 323: 547-555Crossref PubMed Scopus (74) Google Scholar, 16Vincent P. Shareck F. Dupont C. Morosoli R. Kluepfel D. Biochem. J. 1997; 322: 845-852Crossref PubMed Scopus (46) Google Scholar, 17Gielkens M.M.C. Visser J. de Graaf L.H. Curr. Genet. 1997; 31: 22-29Crossref PubMed Scopus (66) Google Scholar, 18Matsuo N. Kaneko S. Kuno A. Kobayashi H. Kusakabe I. Biochem. J. 2000; 346: 9-15Crossref PubMed Scopus (76) Google Scholar, 19Harvey A.J. Hrmova M. De Gori R. Varghese J.N. Fincher G.B. Proteins. 2000; 41: 257-269Crossref PubMed Scopus (100) Google Scholar), and three-dimensional structures (20Zverlov V.V. Liebl W. Bachleitner M. Schwarz W.H. FEMS Microbiol. Lett. 1998; 164: 337-343PubMed Google Scholar). Although most of the characterized enzymes are from saprophytic or rumen microorganisms, several plant α-l-arabinofuranosidases have also been identified. Family 51 arabinoxylan arabinofuranohydrolases, which remove α-l-arabinofuranosyl residues from polymeric arabinoxylans, have been purified from germinated barley grain, and their primary structures have been defined (11Ferré H. Broberg A. Duus J.Ø. Thomsen K.K. Eur. J. Biochem. 2000; 267: 6633-6641Crossref PubMed Scopus (73) Google Scholar, 12Lee R.C. Burton R.A. Hrmova M. Fincher G.B. Biochem. J. 2001; 356: 181-189Crossref PubMed Scopus (84) Google Scholar). There are other reports of the purification or partial purification of higher plant α-l-arabinofuranosidases, but in most cases no amino acid sequence information is available, and it is therefore not possible to classify the enzymes accurately, to draw conclusions about their reaction mechanisms, or to identify their true substrates and biological functions.Here we have purified a bifunctional family 3 α-l-arabinofuranosidase/β-d-xylosidase (ARA-I) from young barley seedlings, defined its kinetic and enzymic properties, and determined its complete amino acid sequence from corresponding cDNAs. The enzyme is unable to hydrolyze arabinoxylans at a significant rate but could play an important role in the complete depolymerization of arabinoxylans through its ability to hydrolyze oligosaccharides released from the polysaccharide by (1 → 4)-β-d-xylan endohydrolases. In parallel, a family 3 β-d-xylosidase (XYL) was purified and characterized.DISCUSSIONAn α-l-arabinofuranosidase and a β-d-xylosidase, both of which are family 3 glycoside hydrolases (13Henrissat B. Biochem. Soc. Trans. 1998; 26: 153-156Crossref PubMed Scopus (150) Google Scholar), were purified ∼1,000-fold (Table I) from extracts of germinated barley grain as outlined in Scheme FS1. The purified enzymes have been designated ARA-I and XYL, respectively. A second, less abundant, α-l-arabinofuranosidase isoenzyme, designated ARA-II, was detected in the extracts (Fig. 1 A) but was not purified to homogeneity. The ARA-I and XYL enzymes have apparent molecular masses of 67 kDa (Fig. 2) and isoelectric points of 5.5 and 6.7, respectively. Examination of their substrate specificities and kinetic properties indicated that α-l-arabinofuranosidase ARA-I can also hydrolyze 4NP-β-d-xylopyranoside, 4NP-β-d-galactopyranoside, and 4NP-α-l-arabinopyranoside at significant rates, whereas the XYL enzyme has a more restricted, or "tighter," specificity for β-d-xylosides (Table III). Thus, the XYL enzyme hydrolyzes not only 4NPX but also, with a 30-fold lower catalytic efficiency, 4NPA. The catalytic efficiency factor for ARA-I was of the same order of magnitude for 4NPA and 4NPX but slightly higher for 4NPA (Table II). For ease of expression we have referred to the enzyme here as an α-l-arabinofuranosidase, but because ARA-I can hydrolyze both substrates efficiently, we acknowledge that it should probably be referred to as a bifunctional α-l-arabinofuranosidase/β-d-xylopyranosidase and that both activities might be important for its biological functionin planta. Certain family 43 (50, 51), 54 (52–54), and 62 (16) α-l-arabinofuranosidases and β-d-xylopyranosidases show similar flexibility in their substrate specificities.Using amino acid sequences generated from the purified barley ARA-I and XYL enzymes, several cDNAs were isolated, and near full-length cDNA sequences were subsequently assembled (Fig. 4). Deduced amino acid sequences indicated that both enzymes have a typical endoplasmic reticulum-targeting signal peptide (Fig. 4) that presumably directs secretion from cells in which they are synthesized. This is a significant observation, given recent indications that the (1 → 4)-β-d-xylan endohydrolase involved in arabinoxylan depolymerization in germinated barley grain is not located in the endomembrane secretory compartment of aleurone layers but is found instead in the cytosol and is likely to be released from aleurone layers only after programmed cell death (56Caspers M.P. Lok F. Sinjorgo K.M. van Zeijl M.J. Nielson K.A. Cameron-Mills V. Plant J. 2001; 26: 191-204Crossref PubMed Scopus (70) Google Scholar, 57Simpson D.J. Fincher G.B. Huang A.H.C. Cameron-Mills V. J. Cereal Sci. 2002; 35: 1-17Crossref Scopus (89) Google Scholar). In isolated aleurone layers, α-l-arabinofuranosidases and β-d-xylosidases are secreted and can be detected in the surrounding medium much earlier than the (1 → 4)-β-xylan endohydrolases (10Banik M. Li C.D. Langridge P. Fincher G.B. Mol. Gen. Genet. 1997; 253: 599-608Crossref PubMed Scopus (54) Google Scholar). Thus, the secretion from aleurone layers of endohydrolases, α-l-arabinofuranosidases, and β-d-xylosidases involved in arabinoxylan degradation is clearly not coincident.Although the NH2 terminus of ARA-I could not be defined with certainty, the cDNAs encode primary translation products of 748 amino acid residues (Fig. 4). The calculated molecular masses of the enzymes, based on these deduced amino acid sequences, are about 80 kDa. This value is much higher than the apparent molecular mass values of 67 kDa observed during SDS-gel electrophoresis of the purified enzymes (Fig. 2) and represents a much longer polypeptide than other plant members of the family 3 group of glycoside hydrolases (19Harvey A.J. Hrmova M. De Gori R. Varghese J.N. Fincher G.B. Proteins. 2000; 41: 257-269Crossref PubMed Scopus (100) Google Scholar). Mass spectrometry was therefore used to examine further the molecular masses of the two enzymes and in both cases confirmed that the enzymes were ∼67 kDa in size. Particular attention was paid to ARA-I, for which the analysis of proteolytic peptides accounted for all regions of the enzyme except the COOH terminus predicted from the cDNA sequence (Fig. 4). The amino acid sequence data also suggested that the COOH termini of the enzymes were heterogeneous and that a single COOH-terminal residue could therefore not be identified. At this stage the weight of evidence suggests that the COOH termini of both ARA-I and XYL are close to the Met-614/Tyr-606 residue of ARA-I/XYL, respectively (Fig. 4). Thus, more than 130 amino acid residues appear to have been removed from the COOH termini during post-translational processing of the enzymes. These values may be compared with the 605 residues found in the family 3 barley β-d-glucan glucohydrolase (58Hrmova M. Harvey A.J. Wang J. Shirley N.J. Jones G.P. Stone B.A. Høj P.B. Fincher G.B. J. Biol. Chem. 1996; 271: 5277-5286Abstract Full Text PDF PubMed Google Scholar). No biological rationale for COOH-terminal processing of the barley ARA-I and XYL enzymes can be provided at this stage. In the case of the barley (1 → 4)-β-d-xylan endohydrolase, both NH2- and COOH-terminal processing of the primary translation product occurs (56Caspers M.P. Lok F. Sinjorgo K.M. van Zeijl M.J. Nielson K.A. Cameron-Mills V. Plant J. 2001; 26: 191-204Crossref PubMed Scopus (70) Google Scholar, 57Simpson D.J. Fincher G.B. Huang A.H.C. Cameron-Mills V. J. Cereal Sci. 2002; 35: 1-17Crossref Scopus (89) Google Scholar).Comparison of the amino acid sequences of the mature enzymes with other members of the family 3 glycoside hydrolases suggested that COOH-terminal processing does not occur in all members of the family. Although β-d-xylosidases from other higher plants are similar in size to those purified here from barley, 2S. Khan, GenBank accession number AC009243, unpublished data. the β-d-xylosidases from Aspergillus niger (59van Peij N.N.M.E. Brinkmann J. Vrsanska M. Visser J. de Graaff L.H. Eur. J. Biochem. 1997; 245: 164-173Crossref PubMed Scopus (102) Google Scholar),Aspergillus oryzae (60Kitamoto N. Yoshino S. Ohmiya K. Tsukagoshi N. Appl. Environ. Microbiol. 1999; 65: 20-24Crossref PubMed Google Scholar), and Erwinia chrysanthemi(61Vroemen S. Heldens J. Boyd C. Henrissat B. Keen N.T. Mol. Gen. Genet. 1995; 246: 465-477Crossref PubMed Scopus (33) Google Scholar) are much larger (∼85 kDa) than the barley enzymes and correspond in size to those predicted from cDNA sequences.The relatively relaxed substrate specificities observed here for the family 3 ARA-I and XYL enzymes from barley can be rationalized in terms of their predicted three-dimensional structures. The three-dimensional structure of a family 3 β-d-glucan glucohydrolase from barley has been solved (62Varghese J.N. Hrmova M. Fincher G.B. Structure. 1999; 7: 179-190Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), and although it is the only family 3 crystal structure available, it has been used to model three-dimensional structures of other family 3 enzymes (19Harvey A.J. Hrmova M. De Gori R. Varghese J.N. Fincher G.B. Proteins. 2000; 41: 257-269Crossref PubMed Scopus (100) Google Scholar). Molecular modeling suggests that the barley ARA-I and XYL enzymes have overall structures similar to that of the β-d-glucan glucohydrolase from barley, although the three-dimensional conformation of the 130-amino acid residue COOH-terminal region of ARA-I and XYL, which is not present in the β-d-glucan glucohydrolase group, cannot be modeled (data not shown).The barley β-d-glucan glucohydrolase has a broad specificity for different linkage types in unsubstituted oligomeric and polymeric β-d-glucan substrates (48Hrmova M. De Gori R. Smith B.J. Fairweather J.K. Driguez H. Varghese J.N. Fincher G.B. Plant Cell. 2002; 14: 1033-1052Crossref PubMed Scopus (82) Google Scholar, 63Hrmova M. Fincher G.B. Carbohydr. Res. 1998; 305: 209-221Crossref Scopus (56) Google Scholar), probably because only two glucosyl residues of the substrate enter the active site pocket and because the glucosyl residue bound at subsite +1 is located between two tryptophan residues that allow some positional flexibility (48Hrmova M. De Gori R. Smith B.J. Fairweather J.K. Driguez H. Varghese J.N. Fincher G.B. Plant Cell. 2002; 14: 1033-1052Crossref PubMed Scopus (82) Google Scholar). The remainder of the substrate projects away from the enzyme surface, and activity is therefore relatively independent of substrate shape and hence of linkage type (34Hrmova M. Varghese J.N. De Gori R. Smith B.J. Driguez H. Fincher G.B. Structure. 2001; 9: 1005-1016Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The barley ARA-I and XYL enzymes examined here also exhibit some flexibility in substrate specificity. Both 4NPA and 4NPX can fit in their catalytic sites. To provide a structural rationale for this observation, the three-dimensional structure of β-d-xylopyranose was taken from the Protein Data Bank and the three-dimensional structure of α-l-arabinofuranose was built. When the two structures were superimposed, a similar stereochemistry was observed about C-1, C-2, and C-3 in both pentoses, and their overall hydrodynamic volumes were also similar (Fig. 7). It is therefore not surprising that the active site of ARA-I can accommodate both substrates.In addition to the aryl glycosides, both enzymes hydrolyze linear oligosaccharides, but neither hydrolyzes substituted polysaccharides. Similarly, the fact that no arabinose is removed from oligoarabinoxylosides released by (1 → 4)-β-d-xylan endohydrolase action (Fig. 3) suggests that neither enzyme is able to hydrolyze substituted oligomeric substrates completely. Only unsubstituted oligoxylosides or oligoarabinoxylosides with two to three unsubstituted xylosyl residues at their nonreducing ends would be expected to fit into a substrate-binding pocket of the shape found in other family 3 enzymes (19Harvey A.J. Hrmova M. De Gori R. Varghese J.N. Fincher G.B. Proteins. 2000; 41: 257-269Crossref PubMed Scopus (100) Google Scholar, 48Hrmova M. De Gori R. Smith B.J. Fairweather J.K. Driguez H. Varghese J.N. Fincher G.B. Plant Cell. 2002; 14: 1033-1052Crossref PubMed Scopus (82) Google Scholar), and only xylose would be released.Family 3 glycoside hydrolases from higher plants can be grouped into two major clades, based on amino acid sequence alignments (48Hrmova M. De Gori R. Smith B.J. Fairweather J.K. Driguez H. Varghese J.N. Fincher G.B. Plant Cell. 2002; 14: 1033-1052Crossref PubMed Scopus (82) Google Scholar). One group contains the broad specificity β-d-glucan glucohydrolases, and the other contains β-d-xylosidases and α-l-arabinofuranosidases. As expected, the ARA-I and XYL enzymes characterized here fall into the second group (data not shown). Although the catalytic amino acid residues, corresponding to Glu-481 and Asp-275 for ARA-I and Glu-474 and Asp-268 for XYL (Fig. 4), are conserved in higher plant family 3 glycoside hydrolases, Hrmovaet al. (48Hrmova M. De Gori R. Smith B.J. Fairweather J.K. Driguez H. Varghese J.N. Fincher G.B. Plant Cell. 2002; 14: 1033-1052Crossref PubMed Scopus (82) Google Scholar) provided a structural explanation for the differences in substrate specificity of the two groups. Thus, the conserved amino acid residue Asp-95 in the β-d-glucan glucohydrolase group that binds the C6-OH of the glycosyl residue bound at subsite −1 is not found in the α-l-arabinofuranosidase/β-d-xylosidase group. Clearly, the pentoses l-arabinofuranose andd-xylose have no C6-OH group, and the α-l-arabinofuranosidase/β-d-xylosidases have a Glu residue in the position corresponding to Asp-95 of the β-d-glucan glucohydrolase group.When the phylogeny of the α-l-arabinofuranosidase/β-d-xylosidase group of family 3 enzymes is examined in more detail (Fig. 8), the higher plant representatives are clearly separated from the fungal representatives. There is one bacterial sequence of Thermotoga neapolitana in this group (Fig. 8). In most cases the true substrate specificities of enzymes encoded by the genes shown in Fig. 8 have not been investigated, and the β-d-xylosidase assignment of identity is based on similarities with a small number of partially characterized enzymes. The dual α-l-arabinofuranosidase/β-d-xylosidase specificity of the barley ARA-I has not been reported for other members of family 3 (13Henrissat B. Biochem. Soc. Trans. 1998; 26: 153-156Crossref PubMed Scopus (150) Google Scholar). It is noteworthy that the barley ARA-I is some distance from XYL in the phylogenetic tree, and this may eventually provide clues for more detailed classification of closely related enzymes in this family.Figure 8Unrooted radial phylogenetic tree of plant family 3 β-d-xylosidases.Amino acid sequences of α-l-arabinofuranosidase/β-d-xylosidase and β-d-xylosidase were aligned with ClustalW (64Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55203) Google Scholar).Branch lengths are drawn to scale. The GenBank accession numbers of the sequences are shown.View Large Image Figure ViewerDownload (PPT)To provide some insight into the likely biological functions of the barley ARA-I and XYL enzymes, expression patterns of the genes were investigated, together with the action of the enzymes on well defined oligomeric and polymeric substrates. Reverse transcription-PCR showed the presence of XYL mRNA in all tissues examined. However, ARA-I mRNA appeared to be absent, or in very low abundance, in the aleurone layer and scutellum of germinated grain (Fig. 5). This is somewhat surprising, given that α-l-arabinofuranosidase activity, measured by activity on 4NPA, has been widely reported in the media surrounding isolated barley aleurone layers (9Taiz L. Honigman W.A. Plant Physiol. 1976; 58: 380-386Crossref PubMed Google Scholar, 10Banik M. Li C.D. Langridge P. Fincher G.B. Mol. Gen. Genet. 1997; 253: 599-608Crossref PubMed Scopus (54) Google Scholar, 65Dashek W.V. Chrispeels M.J. Planta. 1977; 134: 251-256Crossref PubMed Scopus (37) Google Scholar). At the substrate specificity level, XYL was able to hydrolyze (1 → 4)-β-d-xylopentaose to xylose relatively quickly but exhibited no activity against (1 → 5)−α-l-arabinofuranohexaose (Fig. 3,B and C). In contrast, ARA-I hydrolyzed (1 → 5)−α-l-arabinofuranohexaose to arabinose and (1 → 4)-β-d-xylopyranopentaose to xylose, albeit at slow rates (Fig. 3, B and C).Neither enzyme hydrolyzed arabinoxylan at a significant rate, but both ARA-I and XYL rapidly released xylose from oligoarabinoxylosides or oligoxylosides that were first released from the arabinoxylan by the action of (1 → 4)-β-d-xylan endohydrolase. The low levels of arabinose in these hydrolysates (Fig. 3 A) were unexpected, given that this polysaccharide contains about 30% (mol/mol) α-l-arabinofuranosyl residues (55Fincher G.B. Stone B.A. Aust. J. Plant Physiol. 1974; 1: 297-311Crossref Google Scholar), but suggest that neither enzyme can bypass substituted xylosyl residues in oligoarabinoxylosides. In summary, it might be concluded that the ARA-I and XYL enzymes could participate in further hydrolysis of oligosaccharides released from arabinoxylans by endohydrolases in germinated barley grain. The enzymes could also play an important role during cell wall turnover in elongating coleoptiles and in other tissues during normal growth and development. Heteroxylans are major constituents of cell walls in the Poaceae, which include many commercially important cereals and pasture grasses. In the endosperm of barley grains and in elongating coleoptiles, these polysaccharides may comprise 20–70% by weight of the walls (1Fincher G.B. Shewry P.R. Barley: Genetics, Molecular Biology and Bio/Technology. CAB Publishing, Wallingford, U. K.1992: 413-437Google Scholar) and consist of a backbone of (1 → 4)-β-linkedd-xylopyranosyl residues substituted predominantly with α-l-arabinofuranosyl residues. The α-l-arabinofuranosyl residues can be linked to O-3, O-2, or both O-3 and O-2 of xylanopyranosyl residues of the (1 → 4)-β-d-xylan backbone, and other substituents or short side chains are also detected in low abundance (2Viëtor R.J. Kormelink F.J.M. Angelino S.A.G.F. Voragen A.G.J. Carbohydr. Polym. 1994; 24: 113-118Crossref Scopus (35) Google Scholar, 3Carpita N.C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 445-476Crossref PubMed Scopus (681) Google Scholar). The α-l-arabinofuranosyl residues can be esterified with hydroxycinnamic acids, in particular ferulic acid, which may form cross-bridges between adjacent arabinoxylan chains, or with lignin, by oxidative dimerization (4Iiyama K. Lam T.B.T. Stone B.A. Plant Physiol. 1993; 104: 315-320Crossref Scopus (639) Google Scholar). As observed with many wall components in higher plants, the arabinoxylans of primary cell walls can be restructured during normal growth and development. For example, newly synthesized arabinoxylans in maize coleoptiles are deposited in the walls in a highly substituted form, but arabinofuranosyl residues are removed later, and this leads to significant changes in the physicochemical properties of the polysaccharides and hence in the walls themselves (5Andrewartha K. Phillips D.R. Stone B.A. Carbohydr. Res. 1979; 77: 191-204Crossref Scopus (207) Google Scholar, 6Gibeaut D.M. Carpita N.C. Plant Physiol. 1991; 97: 551-561Crossref PubMed Scopus (187) Google Scholar). Removal of α-l-arabinofuranosyl residues is also observed when wall arabinoxylans are degraded (7Wilkie K.C.B. Adv. Carbohydr. Chem. Biochem. 1979; 36: 215-264Crossref Scopus (330) Google Scholar, 8Heyn A.N.J. Plant Sci. 1986; 45: 77-82Crossref Scopus (8) Google Scholar). The presence of α-l-arabinofuranosidases in germinated barley grain or in isolated aleurone layers has been taken as evidence that these enzymes perform this function during the mobilization of the starchy endosperm after cereal grain germination (9Taiz L. Honigman W.A. Plant Physiol. 1976; 58: 380-386Crossref PubMed Google Scholar, 10Banik M. Li C.D. Langridge P. Fincher G.B. Mol. Gen. Genet. 1997; 253: 599-608Crossref PubMed Scopus (54) Google Scholar), although this activity has also been attributed to a separate group of enzymes, known as arabinoxylan α-l-arabinofuranohydrolases (11Ferré H. Broberg A. Duus J.Ø. Thomsen K.K. Eur. J. Biochem. 2000; 267: 6633-6641Crossref PubMed Scopus (73) Google Scholar, 12Lee R.C. Burton R.A. Hrmova M. Fincher G.B. Biochem. J. 2001; 356: 181-189Crossref PubMed Scopus (84) Google Scholar). Most α-l-arabinofuranosidases are so designated because they can hydrolyze the synthetic aryl glycoside 4′-nitrophenyl α-l-arabinofuranoside (4NPA), 1The abbreviations used are: 4NPA, 4′-nitrophenyl α-l-arabinofuranoside; ARA-I, α-l-arabinofuranosidase; HCA, hydrophobic cluster analysis; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; 4NPX, 4′-nitrophenyl β-d-x
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