Bifunctional Family 3 Glycoside Hydrolases from Barley with alpha -L-Arabinofuranosidase and beta -D-Xylosidase Activity

CHARACTERIZATION, PRIMARY STRUCTURES, AND COOH-TERMINAL PROCESSING*

Robert C. LeeDagger, Maria Hrmova, Rachel A. Burton, Jelle Lahnstein, and Geoffrey B. Fincher§

From the Department of Plant Science, University of Adelaide, Waite Campus, Glen Osmond, South Australia 5064, Australia

Received for publication, October 17, 2002, and in revised form, December 1, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

An alpha -L-arabinofuranosidase and a beta -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 alpha -L-arabinofuranoside is only slightly higher than during hydrolysis of 4'-nitrophenyl beta -D-xyloside. Thus, the enzyme is actually a bifunctional alpha -L-arabinofuranosidase/beta -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 beta -D-xyloside, with a catalytic efficiency ~30-fold higher than with 4'-nitrophenyl alpha -L-arabinofuranoside. The enzymes hydrolyze wheat flour arabinoxylan slowly but rapidly hydrolyze oligosaccharide products released from this polysaccharide by (1 right-arrow 4)-beta -D-xylan endohydrolase. Both enzymes hydrolyze (1 right-arrow 4)-beta -D-xylopentaose, and ARA-I can also degrade (1 right-arrow 5)-alpha -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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1) and consist of a backbone of (1 right-arrow 4)-beta -linked D-xylopyranosyl residues substituted predominantly with alpha -L-arabinofuranosyl residues. The alpha -L-arabinofuranosyl residues can be linked to O-3, O-2, or both O-3 and O-2 of xylanopyranosyl residues of the (1 right-arrow 4)-beta -D-xylan backbone, and other substituents or short side chains are also detected in low abundance (2, 3). The alpha -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 (4).

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 (5, 6). Removal of alpha -L-arabinofuranosyl residues is also observed when wall arabinoxylans are degraded (7, 8). The presence of alpha -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 (9, 10), although this activity has also been attributed to a separate group of enzymes, known as arabinoxylan alpha -L-arabinofuranohydrolases (11, 12).

Most alpha -L-arabinofuranosidases are so designated because they can hydrolyze the synthetic aryl glycoside 4'-nitrophenyl alpha -L-arabinofuranoside (4NPA),1 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, alpha -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 (14-19), and three-dimensional structures (20). Although most of the characterized enzymes are from saprophytic or rumen microorganisms, several plant alpha -L-arabinofuranosidases have also been identified. Family 51 arabinoxylan arabinofuranohydrolases, which remove alpha -L-arabinofuranosyl residues from polymeric arabinoxylans, have been purified from germinated barley grain, and their primary structures have been defined (11, 12). There are other reports of the purification or partial purification of higher plant alpha -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 alpha -L-arabinofuranosidase/beta -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 right-arrow 4)-beta -D-xylan endohydrolases. In parallel, a family 3 beta -D-xylosidase (XYL) was purified and characterized.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- DEAE-cellulose (DE52) was from Whatman. CM-Sepharose CL-6B, Polybuffer Exchanger 94, Polybuffer 96, Polybuffer 74, and molecular mass marker proteins were from Amersham Biosciences. Bio-Gel P-100 was from Bio-Rad Laboratories. Phenylmethylsulfonyl fluoride, bovine serum albumin, 4NPA, 4'-nitrophenyl-beta -D-xylopyranoside (4NPX), all other aryl glycosides, laminarin, and arabinogalactan were from Sigma. Coomassie Brilliant Blue reagent was from Pierce, and wheat arabinoxylan, (1 right-arrow 5)-alpha -L-arabinofuranohexaose, (1 right-arrow 4)-beta -D-xylopyranopentaose, and barley (1 right-arrow 3,1 right-arrow 4)-beta -D-glucan were from Megazyme (Bray, Ireland). Larchwood (1 right-arrow 4)-beta -D-xylan and the Thermomyces endoxylanase were kindly provided by Dr. Peter Biely (Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovak Republic).

Enzyme Purification-- Barley (Hordeum vulgare L. cv. Clipper) (3 kg dry weight) was surface sterilized in 0.2% (w/v) AgNO3, washed successively with sterile water, 0.5 M NaCl, and sterile water, and steeped for 24 h in sterile water containing 100 mg/ml chloramphenicol, 100 mg/ml neomycin, 100 units/ml penicillin G, and 100 units/ml nystatin. Germinated grains were maintained at 40-45% (w/w) moisture content by regular application of fresh antibiotic solution for 5 days in the dark at 22 °C. Bacterial or fungal contamination of the grains was not evident at any stage during this period. The germinated barley material was stored overnight at -20 °C prior to homogenization at 4 °C in 1.5 volumes of 0.1 M sodium acetate buffer, pH 5.0, containing 10 mM EDTA, 10 mM sodium azide, 3 mM 2-mercaptoethanol, and 3 mM phenylmethylsulfonyl fluoride. Ammonium sulfate fractional precipitation was performed as described previously (21), and the enzyme purification procedures were as shown in Scheme 1.


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Scheme 1.   Summary of procedures for the purification of ARA-I and XYL.

Enzyme Assays-- Activities of alpha -L-arabinofuranosidase and beta -D-xylosidase were determined spectrophotometrically using 4NPA and 4NPX, respectively. Assays were performed at 37 °C in 50 mM sodium acetate buffer, pH 5.0, containing 4 mM sodium azide and 0.04% (w/v) substrate. Reactions were terminated by the addition of 2 volumes of saturated sodium tetraborate solution. One unit of activity is defined as the amount of enzyme releasing 1 µmol of 4-nitrophenol/min, as measured by absorbance at 410 nm.

Protein Determination and PAGE-- Protein contents of pooled fractions and purified proteins were measured using the Coomassie Brilliant Blue reagent. Purity of column fractions and purified proteins was assessed by SDS-PAGE (22) on 12.5% (w/v) polyacrylamide gels and stained with Coomassie Brilliant Blue R-250.

Substrate Specificities-- Aryl glycosides were used as substrates with purified proteins in standard assays at a final concentration of 2.5 mM. Rates of hydrolysis of polymeric substrates, at a final concentration of 0.2% (w/v), were determined from the increase in reducing sugars (23, 24). Oligosaccharide and monosaccharide products liberated during enzymic hydrolyzes were analyzed by thin layer chromatography on silica gel plates (Merck). Plates were developed in ethyl acetate:acetic acid:water (3:2:1 v/v), and sugars were detected using the orcinol reagent (21).

Kinetic Analyses-- Kinetic parameters of purified enzymes were determined against 4NPA and 4NPX in a concentration range of 0.25-7.0 mM. Assays were performed in triplicate, in 50 mM sodium acetate buffer, pH 4.7, containing 4 mM sodium azide and 160 µg ml-1 bovine serum albumin. S.E. values for assays were less than 5%. Kinetic data were processed by a proportional weighted fit using a nonlinear regression analysis program based on Michaelis-Menten enzyme kinetics (25).

Amino Acid Sequence Analysis-- NH2-terminal sequence analyses of proteins and peptides generated by CNBr, trypsin, or Lys-C and purified by reversed phase HPLC were performed on a Hewlett-Packard G1005A automated protein sequencer, using Edman degradation chemistry. Phenylthiohydantoin-derivatives were identified by reversed phase HPLC using a Hewlett-Packard HPLC 1090 system.

To identify the COOH terminus of ARA-I, the purified enzyme was subjected to extensive amino acid sequence analysis, as follows. First, ARA-I was reduced with 5 mM dithiothreitol, 6 M guanidine HCl in 50 mM Tris-HCl buffer, pH 8.5, at 65 °C for 30 min and alkylated with 20 mM iodoacetamide for 20 min at 20 °C. Dithiothreitol was added to the alkylation solution to remove excess iodoacetamide. Second, the reduced, alkylated protein was fragmented with trypsin or Lys-C. Prior to trypsin digestion, buffer exchange into 100 mM Tris-HCl buffer, pH 7.0, containing 4 M urea and 2 mM dithiothreitol, was performed using a Nanosep Centricon (PALL Life Sciences, Ann Arbor, MI) with a 3-kDa cutoff. After diluting the ARA-1 four times with water, trypsin (Promega) was added for 16 h at 20 °C. For Lys-C digestion, the buffer exchange was into 25 mM Tris-HCl buffer, pH 7.0, containing 4 M urea. Digestion with endoproteinase Lys-C (Promega) was performed for 16 h at 20 °C. The reactions were stopped by adjusting the pH to 3 with 2% (v/v) trifluoroacetic acid.

The Lys-C and tryptic peptides were separated on a Vydac C18 protein column (250 × 2.1 mm, 5 µm; Hesperia, CA). The eluents were (A) (0.05% (v/v) trifluoroacetic acid and (B) 0.04% (v/v) trifluoroacetic acid in 70% (v/v) acetonitrile. The flow rate was 0.2 ml/min, and the gradient was composed of 2-70% (v/v) B for 90 min and 70-100% (v/v) B for an additional 10 min. The column temperature was 40 °C, and protein was detected by absorbance at 214 and 280 nm. Where necessary to separate poorly resolved peptides, selected fractions were rechromatographed using a shallower gradient. Some peptides were fragmented further with 100 mM CNBr in 0.1 M HCl for 16 h at 20 °C.

Mass Spectrometry-- The ARA-I enzyme was desalted by reversed phase HPLC on a 200 × 2.1 mm POROS 20R1 column (Applied Biosystems) with 1% (v/v) formic acid and a 5-100% (v/v) acetonitrile gradient at 0.5 ml min-1 at 40 °C over 15 min. For MALDI-TOF mass spectrometry, desalted ARA-1 and XYL (0.5 µl) mixed with 1 µl of matrix solution (0.1% w/v protein in 30% v/v acetonitrile, 1% v/v formic acid) were spotted onto a target plate and analyzed in a Voyager-DE STR mass spectrometer (Applied Biosystems). Peptide fractions (0.75 µl) were mixed with alpha -cyano-4-hydroxycinnamic acid solution (0.75 µl, 5 g/liter in 50% v/v acetonitrile) and spotted onto a MALDI-TOF mass spectrometry target plate, air-dried, and analyzed in a Voyager-DE STR mass spectrometer. Experimental monoisotopic masses were compared with theoretical peptide masses obtained from the DNA sequence, using the software MS-Digest within the ProteinProspector tool (prospector.ucsf.edu/).

Where no mass was found for a peptide by MALDI-TOF, mass determinations were carried out with an ABI Sciex API 300 electrospray ionization quadrupole mass spectrometer equipped with an ion spray ion source (PE Sciex, Thornhill, Ontario, Canada). Positive ion mass spectra were recorded in a range from m/z 200 to 2,600 and were processed to determine the most probable molecular masses of the peptides using Bio-Multiview software version 1.3 (PE Sciex).

RNA Isolation and PCR-- Total RNA was isolated from root, leaf, coleoptile, and scutellum tissue from barley seedlings, 4 days after germination, using the TRIZOL reagent (Invitrogen) as recommended by the manufacturer. First strand cDNA was prepared from 3 µg of total RNA using the 3'-RACE primer (5'-GACTCGAGTCGACATCGAT17-3') (26) and the THERMOSCRIPT reverse transcriptase system (Invitrogen). cDNA fragments corresponding to sequences predicted from the purified proteins were amplified from the root, shoot, and scutellum single-stranded cDNA preparation by PCR in a mixture containing Taq DNA polymerase, standard PCR buffer, 5 mM dNTPs, 10% (v/v) dimethyl sulfoxide, and 1.5 mM MgCl2, and primed with degenerate oligonucleotide primer pairs, designed on the basis of tryptic peptide sequences. The sequences of these primers were 5'-GGNATHCCNGCNTAYGARTGGTGG-3' (upstream primer) and 5'-AAIGGIGGYTGRAAIGTRTCRTC-3' (downstream primer; I is deoxyriboinosine) corresponding with ARA-I tryptic peptides, and 5'-GTNCCNGCNTAYAAGTGGTGG-3' (upstream primer) and 5'-TCYTGRTTYTGRTCIARICCCAT-3' (downstream primer) corresponding to XYL tryptic peptides. The PCR cycles (35) consisted of a denaturation step (94 °C, 40 s), annealing (50 °C, 40 s), and extension (72 °C, 2 min). Products from the PCR were purified from agarose gels following electrophoresis, and after sequence confirmation, were used to probe lambda ZAP and lambda gt11 cDNA libraries. The 3'-ends of the ARA-I and XYL cDNAs were amplified using the 3'-end RACE PCR procedure (26); gene-specific primers were based on cDNA sequences of partial cDNA clones that were isolated from cDNA libraries. The ARA-I 3'-end cDNA was amplified by two successive rounds of PCR (98 °C, 40 s; 50 °C, 40 s; 72 °C, 3 min) with root/shoot/scutellum cDNA, 3'-RACE adaptor primer 5'-GACTCGAGTCGACATCG-3', and gene-specific oligonucleotide primers 5'-CGGCGTACGAGTGGTGGTCCGAAG-3' (round 1) and 5'-CGCTGCACGGCGTGTCATACGT-3' (round 2). The 3'-end of the XYL cDNA was isolated using a single round of PCR, as described for ARA-I, with the XYL-specific primer, 5'-GGATACATCACGTCGGAC-3'. The largest amplified products were purified from agarose gels, ligated into the pGEM TEasy vector (Promega) using T4 DNA ligase (New England BioLabs), and introduced into competent DH5-alpha cells by electroporation using the Bio-Rad Gene-Pulser apparatus.

cDNA Isolation-- A lambda ZAP-cDNA library (Stratagene, La Jolla, CA) was prepared from poly(A)+ RNA of 24-48 h gibberellic acid3-treated barley (cv. Clipper) aleurone layers, and a lambda gt11 library was prepared from 12-day-old barley (cv. Klages) seedlings (Clontech). The libraries were screened on nitrocellulose membranes (Micron Separations Inc., Westborough, MA). Plaque replicas were hybridized with [alpha -32P]dCTP-labeled ARA-I cDNA (500 bp) and XYL cDNA (1,300 bp) fragments as described (27). Positive clones were identified by autoradiography and purified by further rounds of screening. The cDNA inserts were excised from the Uni-ZAP XR vector (lambda ZAP clone) or, in the case of lambda gt11 clones, subcloned into the pBluescript SK(+) vector (Stratagene).

Nucleotide Sequence Analysis-- Both strands of isolated cDNAs were sequenced using the dideoxynucleotide chain termination procedure (28). Data from automated sequencing were compiled and analyzed using the Seq-Ed program (Applied Biosystems), and further analyses of DNA sequences and data base searches were performed using the University of Wisconsin Genetics Computer Group software (29) in the ANGIS suite of programs at the University of Sydney (www.angis.org.au/WebANGIS/). Sequences were aligned using the ClustalW program (www2.ebi.ac.uk/clustalw).

Reverse Transcription-PCR-- cDNA samples were prepared from 3 µg of total RNA from developing grains (6 days postanthesis), leaf (5 days after germination), coleoptile (4 days), rootlets (5 days), aleurone (3 days), and scutella (3 days). Reverse transcriptase reactions were primed with (dT)17 primer. PCRs contained 1 µl of cDNA and 0.1 µg of each gene-specific primer. DNA fragments corresponding to each gene were amplified by 30 cycles of PCR consisting of 94 °C, 40 s; 55 °C, 40 s; 72 °C, 30 s. Amplified products were detected by gel electrophoresis, and DNA bands were observed under ultraviolet light. For each set of primers, amplified DNA was excised from the agarose gel and purified for subsequent DNA sequence analysis. DNA sequences, in every case, exactly matched the DNA sequence of the respective cDNA clone.

Genetic Mapping of ARA-I and XYL Genes-- Barley mapping populations and parental lines were screened at high stringency with probes corresponding to each of the XYL and ARA-I cDNAs, as described (30). The DNA probe for ARA-I was the 2,400-bp 3'-end RACE fragment, and for XYL was the 1,800-bp 3'-end RACE fragment. Filters were hybridized with radiolabeled DNA probes at 65 °C and washed in 0.1 × SSC, 0.1% (w/v) SDS at 65 °C to remove nonspecifically bound probe DNA. Chromosomal locations for the ARA-I and XYL genes were allocated by correlation with genetic markers using the Mapmaker and JoinMap software (31, 32).

Hydrophobic Cluster Analysis and Molecular Modeling-- Hydrophobic cluster analysis (HCA) was performed using DrawHCA (33; www.lmcp.jussieu.fr/~callebau/hca_method.html). The three-dimensional molecular models of ARA-I and XYL were built using the three-dimensional structure of barley beta -D-glucan glucohydrolase (www.rcsb.org/pdb; PDB accession code 1IEQ; 34) as a template, and Modeler, version 6.1 (35). The models were checked manually on O (36) and evaluated using PROCHECK (37) and PROSAII (38). The three-dimensional structure of alpha -L-arabinofuranose was built and minimized in SYBYL 6.62 (39), and molecular surfaces of beta -D-xylopyranose (PDB accession code 1B3V) and alpha -L-arabinofuranose were computed in GRASP (40), using a probe size of 1.4 Å and all atoms having fixed radii.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Purification of Barley ARA-I and XYL-- Preliminary experiments showed that alpha -L-arabinofuranosidase and beta -D-xylosidase activities in young barley seedlings reached a peak 4-5 days after germination. From 3 kg of germinated barley grain, an extract of soluble proteins from 5-day-old barley seedlings yielded 45 and 46 units of 4NPA- and 4NPX-hydrolyzing activities, respectively (Table I). The steps used for the purification of the ARA-I and XYL isoenzymes are summarized in Scheme 1.

                              
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Table I
Enzyme yields and purification factors of ARA-I and XYL

Two peaks of alpha -L-arabinofuranosidase activity were resolved on the DEAE-cellulose column at ~90 mM NaCl and 140 mM NaCl (Fig. 1A) and were designated ARA-I and ARA-II, respectively. Attempts to purify ARA-II completely were unsuccessful, and this isoenzyme will not be described here. The ARA-I fractions always contained beta -D-xylosidase activity at ~15% of their alpha -L-arabinofuranosidase activity, throughout the purification of ARA-I (Fig. 1B) and in the final purified enzyme preparation. It appeared therefore that although ARA-I had a preference for 4NPA, it could also hydrolyze 4NPX. The final ARA-I preparation was purified 1,080-fold and represented about 2% of the initial activity (Table I). It should be noted that the true purification factor of ARA-I was likely to be significantly higher than 1,080-fold because of the ARA-II present in the initial tissue extracts. The XYL enzyme was separated from ARA-I during the initial DEAE-cellulose chromatography step (Scheme 1) and, after resolution from other proteins on CM-cellulose (Fig. 1C), was ultimately purified 960-fold (Table I).


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Fig. 1.   Chromatography of the 40-60% (NH4)2SO4-insoluble fraction of barley seedling extracts. A, material precipitated at 40-60% saturated (NH4)2SO4 was applied to DEAE-cellulose at pH 8.2, and bound proteins were eluted with a 2-liter gradient of 0-350 mM NaCl. Fractions (20 ml) were assayed against 4NPA (black-triangle) and 4NPX (triangle ) and protein (-). B, fractions 21-29 from DEAE-cellulose were pooled and applied to CM-Sepharose at pH 4.2. Bound proteins were eluted with an 800-ml gradient of 0-400 mM NaCl, and fractions were assayed as described for DEAE-cellulose. C, material not bound to DEAE-cellulose was concentrated and applied to CM-Sepharose at pH 5.0. Bound protein was eluted with a 1.6-liter gradient of 0-400 mM NaCl.

In view of the apparent activity of the ARA-I on both 4NPA and 4NPX during the purification process, particular care was taken to evaluate the purities of the final enzyme preparations. The final ARA-I preparation appeared as a single protein of molecular mass 67 kDa after SDS-gel electrophoresis (Fig. 2A) and had an isoelectric point of 5.5. Although the NH2-terminal sequence could not be obtained with ARA-I, presumably because the NH2-terminal residue was blocked to Edman degradation, the sequences of 10 tryptic peptides from the enzyme were determined. For the final XYL preparation, which had a molecular mass of 67 kDa (Fig. 2B) and an isoelectric point of 6.7, a minor protein band greater than 100 kDa could be seen but was not resolved by additional chromatography steps (data not shown). An NH2-terminal sequence of 38 amino acid residues was obtained for XYL, together with the sequences of six tryptic peptides. For both ARA-I and XYL, amino acid sequence comparisons with protein sequences in the data bases revealed that the enzymes were members of the family 3 group of glycoside hydrolases (13).


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Fig. 2.   SDS-PAGE of protein fractions during the purification of ARA-I and XYL. A: lane 1, molecular mass markers; lane 2, seedling homogenate; lane 3, 40-60% (NH4)2SO4 fraction; lane 4, DEAE-cellulose ARA-I peak (fractions 21-29); lane 5, CM-Sepharose pooled fractions 10-14; lane 6, Polybuffer Exchanger 94 fractions; lane 7, purified ARA-I after Bio-Gel P-100. B: lanes 1-3, as for A above; lane 4, DEAE-cellulose unbound fraction; lane 5, CM-Sepharose pooled fractions 39-46; lane 6, Polybuffer Exchanger 94 fractions; lane 7, purified XYL after Bio-Gel P-100.

Kinetic Analyses-- The kinetic parameters for ARA-I and XYL using 4NPA and 4NPX, respectively, are shown in Table II. The Km values for ARA-I against aryl glycosides revealed that this enzyme had a relatively low Km for 4NPX compared with 4NPA, although the catalytic rate was higher on 4NPA (Table II).

                              
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Table II
Kinetic parameters of ARA-I and XYL

Comparison of kinetic parameters for XYL against 4NPA and 4NPX demonstrated a definite preference for 4NPX (Table II). XYL had a relatively low Km value for 4NPX (1.7 ± 0.04 mM) compared with the Km value for 4NPA (24.8 ± 0.04 mM), the catalytic rate constant for 4NPX was twice that for 4NPA, and the catalytic efficiency factor for 4NPX was about 30 times that measured for 4NPA (Table II).

Substrate Specificities-- The pH optimum for both ARA-I and XYL was 4.7 (data not shown). The preferred aryl glycoside substrates for ARA-I and XYL were 4NPA and 4NPX, respectively. ARA-I could also hydrolyze 4NPX, 4NP-beta -D-galactopyranoside, and 4NP-alpha -L-arabinopyranoside, with 20, 16, and 11% of the specific activity observed for 4NPA, respectively (Table III). The preference of XYL for 4NPX was more pronounced, with specific activities for other aryl glycosides no greater than 3% of that for 4NPX (Table III).

                              
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Table III
Relative activities of ARA-I and XYL against aryl glycosides

Of the polysaccharides examined, ARA-I and XYL hydrolyzed only arabinoxylan, but hydrolytic rates were very low, and activity could be detected only after prolonged incubation of the enzymes with this substrate (Fig. 3A). When ARA-I was incubated with the arabinoxylan, small amounts of both L-arabinose and D-xylose were released, but XYL released only D-xylose (Fig. 3A). Neither enzyme hydrolyzed larch arabinogalactan, (1 right-arrow 3,1 right-arrow 4)-beta -D-glucan, laminarin, or CM-xylan (data not shown).


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Fig. 3.   Substrate specificities of ARA-I and XYL. A, thin layer chromatography of hydrolytic products released by ARA-I and XYL (first and second lanes, respectively) from wheat flour arabinoxylan. The third lane shows products released from the arabinoxylan by the Thermomyces endoxylanase. Products of the endoxylanase mixed with ARA-I or XYL are shown in the fourth and fifth lanes, respectively. Standards were L-arabinofuranose, D-xylopyranose, and oligoxylopyranosides xylobiose-xylohexaose (sixth, seventh, and eighth lanes, respectively). B, products released when ARA-I and XYL were incubated with xylopyranopentaose after 0.5, 10, and 60 min. C, products released when ARA-I and XYL were incubated with (1right-arrow5)-alpha -L-linked arabinofuranohexaose for 10 and 60 min.

In the presence of (1 right-arrow 4)-beta -D-xylan endohydrolase, hydrolysis rates increased dramatically, and both ARA-I and XYL released large amounts of D-xylose; ARA-I also released some L-arabinose (Fig. 3A). In both cases, ARA-I or XYL was added at a concentration 1/10 of that added in assays without the addition of the (1 right-arrow 4)-beta -D-xylan endohydrolase. Thus, the partial endohydrolysis of the arabinoxylan greatly enhanced the release of oligosaccharides and monosaccharides from this substrate (Fig. 3A). XYL completely hydrolyzed xylopyranopentaose to D-xylose within 1 h. ARA-I was less efficient in hydrolyzing xylopyranopentaose and after 1 h, had degraded the substrate to approximately the same degree as XYL after 10 min (Fig. 3B). (1 right-arrow 5)-alpha -L-Arabinofuranohexaose was only partially degraded by ARA-I in 1 h, and XYL did not hydrolyze this substrate to any significant extent (Fig. 3C).

Isolation of cDNAs-- Degenerate oligonucleotide primers were designed to correspond with ARA-I tryptic amino acid sequences, and PCR was performed using all combinations of forward and reverse primers with reverse transcribed, 3-day-old barley seedling mRNA. The nucleotide sequence of a 500-bp PCR product corresponded exactly with the amino acid sequences of various tryptic peptides from purified ARA-I, and the PCR product was therefore used to probe a cDNA library from 24-48 h gibberellic acid3-induced aleurone layers. Of 200,000 cDNA clones screened, a single 1,900-bp ARA-I cDNA was identified. The missing 5'-end fragment of the cDNA was subsequently isolated during additional screening of the cDNA library, and 3'-RACE was used to generate the missing 3'-end fragment. The fragments were used to assemble the full nucleotide sequence of the ARA-I cDNA, and a similar strategy was used to assemble a near full-length cDNA encoding the XYL enzyme (data not shown). In both cases a strong bias in codon usage was evident for the two genes, with G or C residues found in the wobble base position of about 95% of ARA-I and XYL codons in the region encoding the mature enzymes (data not shown).

Primary Structures of the Enzymes-- The complete amino acid sequences of the barley ARA-I and XYL enzymes were deduced from the nucleotide sequences of the corresponding cDNAs (Fig. 4). For the XYL enzyme, 38 amino acids were sequenced from the NH2 terminus of the purified enzyme. This showed that the NH2-terminal residue of the mature enzyme was the Ala residue of the ADPPF sequence indicated in Fig. 4. The sequence of the 38 NH2-terminal amino acid residues corresponded with the sequence deduced from the cDNA in 31 positions, whereas the sequences of another 168 amino acids derived from 8 tryptic peptides matched exactly with the sequence deduced from the cDNA. The fact that the experimentally determined and deduced NH2-terminal sequences did not exactly match raised the possibility that the cDNA sequence was in fact a composite sequence of genes encoding two closely related XYL isoenzymes from the barley cDNA libraries. However, the overlapping region of the two 5' cDNA fragments was 861 bp in length (Fig. 4), and the overlapping nucleotide sequences of the various cDNA fragments matched exactly. At this stage we are unable to explain this apparent discrepancy.


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Fig. 4.   Alignment of ARA-I and XYL amino acid sequences. Identical residues in both sequences are shown in red. The pairwise alignment was prepared using ClustalX (41). The ARA-I and XYL cDNAs encode mature polypeptides of 748 amino acid residues with signal peptides of 29 residues each (shading). The amino acid residues are numbered from the NH2-terminal amino acid residues (arrowheads) of the mature enzymes. An arrowhead is also used to indicate the likely COOH terminus of ARA-1, and asterisks indicate potential N-glycosylation sites (marked as CHO). Arrows indicate the putative catalytic nucleophiles (Asp-275 for ARA-I and Asp-268 for XYL) and putative catalytic acid/bases (Glu-481 for ARA-I and Glu-474 for XYL). Blue overlines above the sequence of ARA-1 indicate the amino acid sequences that were confirmed by either NH2-terminal or peptide sequencing with MALDI-TOF or electrospray ionization quadrupole mass spectrometry analyses, after proteolytic cleavage by Lys-C, trypsin, or CNBr.

No NH2-terminal sequence was obtained for the purified ARA-I enzyme, although the sequences of a total of 126 amino acid residues from 9 different tryptic peptides matched the sequence deduced from the cDNA fragments (Fig. 4). A few amino acid differences were observed, but these were attributed to differences in the varieties used to isolate the enzyme and the cDNA library. The NH2-terminal sequence of ARA-I (AEAQAQAPVF) was predicted using the SigCleave program and corresponded to the experimentally determined sequence for the NH2 terminus of the XYL enzyme (Fig. 4). In both cases the -3, -1 rule of von Heijne (42) was satisfied, but there was no obvious reason why the NH2-terminal residue of ARA-I would be blocked to Edman degradation (43).

If it is accepted that the NH2-terminal residue of ARA-I is as indicated in Fig. 4, the ARA-I and XYL cDNAs both encode mature polypeptides of 748 amino acids, and these show 51% positional identity for the two enzymes. Signal peptides of 29 residues (Fig. 4) were detected for each enzyme, and these have characteristics typical of those from other eukaryotic signal peptides that direct nascent polypeptides to the endoplasmic reticulum (44). The molecular mass calculated from the deduced amino acid sequence was 79,184 Da for ARA-I, which had a calculated isoelectric point of 5.7. The corresponding values calculated for XYL were 80,500 Da and 6.5 for XYL, respectively. Although the calculated isoelectric point values correspond well with the values of 5.5 and 6.7 for ARA-I and XYL, respectively, as determined from the purified enzymes, the molecular masses deduced from the cDNA sequences are considerably higher than the values of 67 kDa obtained for the purified enzymes on SDS gels run under reducing conditions (Fig. 2). This would suggest that processing of the primary translation product might have occurred and that a peptide fragment of up to about 12 kDa might have been removed during enzyme maturation. However, the NH2-terminal end of XYL has certainly not been processed, and the presence of a Lys-C peptide starting at residue 29 of ARA-I suggests that a fragment of 12 kDa has not been cleaved from the NH2 terminus of this enzyme either (Fig. 4). Internal processing of the enzymes would also be possible, but the 5 mM 2-mercaptoethanol included here in the gel loading buffer during electrophoresis and during enzyme purification would dissociate individual peptide chains linked by disulfide bonds (45), and we could find no evidence for internal processing of the two enzymes.

In view of the discrepancy between observed and predicted apparent molecular mass values, ARA-I and XYL were examined by MALDI-TOF mass spectrometry. Broad peaks of 69.4 kDa and 68.2 kDa, with widths of 3-5 kDa, were obtained for ARA-I and XYL, respectively (data not shown). The amino acid sequence of ARA-I was investigated in more detail. Using a combination of MALDI-TOF and electrospray ionization mass spectrometry of tryptic and Lys-C peptides, coupled with Edman sequence analysis of the peptides, most of the NH2-terminal and central peptides predicted from the ARA-I cDNA sequence could be identified (Fig. 4). However, the COOH-terminal region of the enzyme predicted by the cDNA sequence was not detected in any of the enzymic digests, and it was concluded on this basis and on the basis of domain prediction (46) that the actual COOH terminus of the mature ARA-I enzyme is in the vicinity of Met-614 (Fig. 4).

Catalytic Amino Acid Residues and Active Sites-- Based on multiple sequence alignments (47) and HCA (33), the putative catalytic nucleophiles are predicted to be Asp-275 for ARA-I and Asp-268 for XYL (Fig. 4). These residues are absolutely conserved in family 3 glycoside hydrolases (19). Prediction of the catalytic acid/base residues of the enzymes is somewhat more complicated. Multiple sequence alignments and HCA clearly identify two candidate amino acid residues for this role (data not shown). Hrmova et al. (48) suggested that the catalytic acid/base for beta -D-xylosidase-like members of the family 3 group would correspond to Glu-479 for ARA-I and Glu-472 for XYL. However, at this stage we believe that the catalytic residues could just as easily be Glu-481 for ARA-I and Glu-474 for XYL (Fig. 4). Molecular modeling experiments, in which the known three-dimensional structure of the barley beta -D-glucan glucohydrolase is used as a template (35), indicate that the Glu-481 residue is more appropriately positioned with respect to the known catalytic acid/base residue Glu-491 of the beta -D-glucan glucohydrolase (data not shown). In these models the catalytic acid/base and nucleophile amino acid residues are about 6.5 Å apart. However, it must be pointed out that the sequence identities of the template and target enzymes are ~30% and that this is considered in the "twilight zone" of reliability of the molecular modeling programs (49).

Other features of family 3 glycoside hydrolases which can be observed in the ARA-I and XYL sequences include the conserved WGR and KH motifs, beginning at residues Trp-147 and Lys-192 for ARA-I, and Trp-139 and Lys-185 for XYL. These motifs are probably involved in substrate binding (34, 48). In addition, sequences similar to the conserved COOH-terminal antiparallel loop of family 3 enzymes (34) were present in the region starting at about residue 559 in ARA-I and 562 in XYL (Fig. 4).

Expression Analysis of ARA-I and XYL Genes-- Transcript levels of ARA-I and XYL in various barley tissues, including developing grain, aleurone, scutellum, rootlets, coleoptiles, and leaves, both etiolated and light-grown, were assessed by Northern hybridization analyses, but hybridization signals were very low. Gene-specific primers were therefore synthesized to amplify specifically short DNA fragments, using reversed transcription PCR, from reverse transcribed total RNA from each plant tissue sample. Sense oligonucleotide primers corresponded to cDNA sequences within the coding region of respective cDNAs, and antisense primers were designed for sequences within the unique 3'-untranslated regions of respective cDNAs.

ARA-I transcripts were detected in developing grains and in the vegetative tissues of rootlets, coleoptiles, and leaves (Fig. 5). ARA-I does not appear to be transcribed in aleurone or scutellum tissue 3 days after germination. XYL transcripts were found in each of the barley tissues and at relatively high levels (Fig. 5).


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Fig. 5.   Reverse transcription-PCR analysis for ARA-I and XYL. DNA was amplified from total RNA preparations of developing barley grain (DG), aleurone (Al), scutellum (Sc), root (R), coleoptile (C), etiolated leaf (Let), and light-grown leaf (Llg), using ARA-I (A) and XYL (B) gene-specific primers. M indicates molecular markers, and C is a control, in which no cDNA was present.

Genetic Mapping of ARA-I and XYL Genes-- Single dominant bands were evident when Southern hybridization analyses of parental lines and mapping population DNA samples were probed with ARA-I and XYL cDNAs (data not shown). This suggested that single genes encoding these enzymes are present on the barley genome, although it should be noted that the hybridizations were performed at high stringency to avoid cross-hybridization between the ARA-I and XYL probes, which are about 50% identical, and related genes might therefore have gone undetected. Indeed, preliminary amino acid sequence analysis of a protein band enriched in ARA-II revealed differences with ARA-I sequences. Despite the fact that ARA-II was not completely purified, it could be concluded from the sequence comparisons that there are at least two genes encoding alpha -L-arabinofuranosidases in barley (data not shown).

Restriction fragment length polymorphisms (RFLPs) for the DNA probes were rare, with only one RFLP for ARA-I found in DNA digested with HindIII from the parents Chebec and Harrington and for XYL RFLPs were present only for Clipper and Sahara genomic DNA digested with EcoRI or DraI. The ARA-I gene is located on the long arm of barley chromosome 2H, between the molecular markers ABC165 and BCD512, and XYL is found near the centromere of barley chromosome 6H, between markers Bmag9 and BCD269 (Fig. 6).


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Fig. 6.   The positions of the ARA-I and XYL genes on barley chromosomes 2H and 6H. A, ARA-I was mapped using a Chebec and Harrington mapping population. ARA-I is indicated with an arrow. B, XYL was mapped using a Clipper and Sahara mapping population. Xyl is indicated with an arrow.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

An alpha -L-arabinofuranosidase and a beta -D-xylosidase, both of which are family 3 glycoside hydrolases (13), were purified ~1,000-fold (Table I) from extracts of germinated barley grain as outlined in Scheme 1. The purified enzymes have been designated ARA-I and XYL, respectively. A second, less abundant, alpha -L-arabinofuranosidase isoenzyme, designated ARA-II, was detected in the extracts (Fig. 1A) 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 alpha -L-arabinofuranosidase ARA-I can also hydrolyze 4NP-beta -D-xylopyranoside, 4NP-beta -D-galactopyranoside, and 4NP-alpha -L-arabinopyranoside at significant rates, whereas the XYL enzyme has a more restricted, or "tighter," specificity for beta -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 alpha -L-arabinofuranosidase, but because ARA-I can hydrolyze both substrates efficiently, we acknowledge that it should probably be referred to as a bifunctional alpha -L-arabinofuranosidase/beta -D-xylopyranosidase and that both activities might be important for its biological function in planta. Certain family 43 (50, 51), 54 (52-54), and 62 (16) alpha -L-arabinofuranosidases and beta -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 right-arrow 4)-beta -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 (56, 57). In isolated aleurone layers, alpha -L-arabinofuranosidases and beta -D-xylosidases are secreted and can be detected in the surrounding medium much earlier than the (1 right-arrow 4)-beta -xylan endohydrolases (10). Thus, the secretion from aleurone layers of endohydrolases, alpha -L-arabinofuranosidases, and beta -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 (19). 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 beta -D-glucan glucohydrolase (58). 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 right-arrow 4)-beta -D-xylan endohydrolase, both NH2- and COOH-terminal processing of the primary translation product occurs (56, 57).

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 beta -D-xylosidases from other higher plants are similar in size to those purified here from barley,2 the beta -D-xylosidases from Aspergillus niger (59), Aspergillus oryzae (60), and Erwinia chrysanthemi (61) 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 beta -D-glucan glucohydrolase from barley has been solved (62), 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 (19). Molecular modeling suggests that the barley ARA-I and XYL enzymes have overall structures similar to that of the beta -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 beta -D-glucan glucohydrolase group, cannot be modeled (data not shown).

The barley beta -D-glucan glucohydrolase has a broad specificity for different linkage types in unsubstituted oligomeric and polymeric beta -D-glucan substrates (48, 63), 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 (48). 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 (34). 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 beta -D-xylopyranose was taken from the Protein Data Bank and the three-dimensional structure of alpha -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.


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Fig. 7.   Comparison of three-dimensional conformations of beta -D-xylopyranose and alpha -L-arabinofuranose. The two stereochemically related pentoses beta -D-xylopyranose and alpha -L-arabinofuranose (in cpk colors) are superimposed and show the spatial coincidence of C-1, C-2, and C-3 of the two pentoses. Molecular surfaces of beta -D-xylopyranose and alpha -L-arabinofuranose are colored in white and black, respectively, and are indicative of the hydrodynamic volumes of the molecules. The image was generated using GRASP (40).

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 right-arrow 4)-beta -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 (19, 48), 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 (48). One group contains the broad specificity beta -D-glucan glucohydrolases, and the other contains beta -D-xylosidases and alpha -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, Hrmova et al. (48) provided a structural explanation for the differences in substrate specificity of the two groups. Thus, the conserved amino acid residue Asp-95 in the beta -D-glucan glucohydrolase group that binds the C6-OH of the glycosyl residue bound at subsite -1 is not found in the alpha -L-arabinofuranosidase/beta -D-xylosidase group. Clearly, the pentoses L-arabinofuranose and D-xylose have no C6-OH group, and the alpha -L-arabinofuranosidase/beta -D-xylosidases have a Glu residue in the position corresponding to Asp-95 of the beta -D-glucan glucohydrolase group.

When the phylogeny of the alpha -L-arabinofuranosidase/beta -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 beta -D-xylosidase assignment of identity is based on similarities with a small number of partially characterized enzymes. The dual alpha -L-arabinofuranosidase/beta -D-xylosidase specificity of the barley ARA-I has not been reported for other members of family 3 (13). 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.


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Fig. 8.   Unrooted radial phylogenetic tree of plant family 3 beta -D-xylosidases. Amino acid sequences of alpha -L-arabinofuranosidase/beta -D-xylosidase and beta -D-xylosidase were aligned with ClustalW (64). Branch lengths are drawn to scale. The GenBank accession numbers of the sequences are shown.

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 alpha -L-arabinofuranosidase activity, measured by activity on 4NPA, has been widely reported in the media surrounding isolated barley aleurone layers (9, 10, 65). At the substrate specificity level, XYL was able to hydrolyze (1 right-arrow 4)-beta -D-xylopentaose to xylose relatively quickly but exhibited no activity against (1 right-arrow 5)-alpha -L-arabinofuranohexaose (Fig. 3, B and C). In contrast, ARA-I hydrolyzed (1 right-arrow 5)-alpha -L-arabinofuranohexaose to arabinose and (1 right-arrow 4)-beta -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 right-arrow 4)-beta -D-xylan endohydrolase. The low levels of arabinose in these hydrolysates (Fig. 3A) were unexpected, given that this polysaccharide contains about 30% (mol/mol) alpha -L-arabinofuranosyl residues (55), 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.

    ACKNOWLEDGEMENTS

We thank Dr. Peilin Xu for providing the aleurone cDNA library, Dr. Ken Chalmers for assistance with the genetic mapping, Dr. Neil Shirley for help with the protein analyses, and Dr. Yoji Hayasaka and Kris Ferguson for elements of the mass spectrometric analyses. Professor Bruce Stone, Dr. Hugues Driguez, Dr. Ross De Gori, and Dr. Andrew Harvey provided valuable advice in various aspects of the project.

    FOOTNOTES

* This work was supported in part by grants from the Australian Research Council and the Grains Research and Development Corporation of Australia (to G. B. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY029259 (ARA-I) and AY029260 (XYL).

Dagger Recipient of a postgraduate scholarship from the Grains Research and Development Corporation of Australia.

§ To whom correspondence should be addressed. Tel.: 61-8-8303-7296; Fax: 61-8-8303-7109; E-mail: geoff.fincher@adelaide.edu.au.

Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M210627200

2 S. Khan, GenBank accession number AC009243, unpublished data.

    ABBREVIATIONS

The abbreviations used are: 4NPA, 4'-nitrophenyl alpha -L-arabinofuranoside; ARA-I, alpha -L-arabinofuranosidase; HCA, hydrophobic cluster analysis; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; 4NPX, 4'-nitrophenyl beta -D-xylopyranoside; RACE, rapid amplification of cDNA ends; RFLP, restriction fragment length polymorphism; XYL, beta -D-xylosidase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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