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. 2004 Jun;135(2):867-78.
doi: 10.1104/pp.104.041269. Epub 2004 Jun 4.

Purification and characterization of enzymes exhibiting beta-D-xylosidase activities in stem tissues of Arabidopsis

Zoran Minic  1 , Christophe Rihouey, Cao Trung Do, Patrice Lerouge, Lise Jouanin
Affiliations

Purification and characterization of enzymes exhibiting beta-D-xylosidase activities in stem tissues of Arabidopsis

Zoran Minic et al. Plant Physiol. 2004 Jun.

Abstract

This work describes the purification and characterization of enzymes that exhibit beta-d-xylosidase activity in stem tissues of Arabidopsis. This is the first detailed investigation that concerns the characterization of catalytic properties and sequence identity of enzymes with beta-D-xylosidase activities in a dicotyledonous plant. Three different enzymes, ARAf, XYL4, and XYL1 with apparent molecular masses of 75, 67, and 64 kD, respectively, were purified to homogeneity. ARAf was identified as a putative alpha-L-arabinofuranosidase, and XYL4 and XYL1 as putative beta-D-xylosidases using matrix-assisted laser-desorption ionization time of flight. ARAf belongs to family 51 and XYL4 and XYL1 to family 3 of glycoside hydrolases. ARAf and XYL1 have highest specificity for p-nitrophenyl-alpha-L-arabinofuranoside and XYL4 for p-nitrophenyl-beta-D-xylopyranoside and natural substrates such as xylobiose and xylotetraose. XYL4 was shown to release mainly D-Xyl from oat spelt xylan, rye arabinoxylan, wheat arabinoxylan, and oligoarabinoxylans. ARAf and XYL1 can also release D-Xyl from these substrates but less efficiently than XYL4. Moreover, they can also release L-Ara from arabinoxylans and arabinan. Overall, the results indicate that XYL4 possesses enzymatic specificity characteristic for a beta-D-xylosidase, while ARAf and XYL1 act as bifunctional alpha-L-arabinofuranosidase/beta-D-xylosidases. Analysis of the activity of these three enzymes in stem tissues at different stages of development has shown that young stems possess the highest activities for all three enzymes in comparison to the activities of the enzymes present in stems at older stages of development. High enzyme activities are most likely related to the necessary modifications of cell wall structure occurring during plant growth.

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Figures

Figure 1.
Figure 1.
Chromatographic purification of enzymes exhibiting β-d-xylosidase activity from stem tissues of Arabidopsis (see also Table I). Purification Step 1, A 1-mL sample of stem tissue cell-free extract (2.5 mg proteins) was loaded on a 0.5- × 3-cm column of Con A Sepharose. The column was eluted with 7 mL of 20 mm Tris-HCl (pH 7.4), 0.5 m NaCl, and 0.2 m methyl-α-glucopyranoside buffer. Fractions of 1 mL were collected, and aliquots of 50 μL were assayed for β-d-xylosidase activity. Purification Step 2, Pooled fractions from the first Con A Sepharose chromatography were dialysed, concentrated, and loaded on a CM-Trisacryl cation-exchange column (1.5 × 5 cm). The column was eluted discontinuously in 15 steps with 2-mL fractions of 25 mm Na-acetate, pH 5.0, and 0.015% Triton X-100, containing increasing concentrations of NaCl from 0.0 to 5.0 m. Fractions of 1 mL were collected, and aliquots of 100 μL were assayed for β-d-xylosidase activity. Step 3, Gel-filtration chromatography. The pooled fractions showing β-d-xylosidase activity were concentrated to 500 μL and loaded on a Superdex 200 HR10/30 column (Amersham Pharmacia Biotech). Elution was performed at room temperature with 20 mm Na-acetate buffer (pH 5.0) containing 150 mm NaCl. Fractions of 0.4 mL were collected at a flow rate of 0.5 mL/min, and 100 μL of each fraction was assayed for β-d-xylosidase activity as described in “Materials and Methods.”
Figure 2.
Figure 2.
SDS-PAGE of purified enzymes. The purified peak fractions from Figure 1 containing about 2 μg of protein were analyzed by SDS-PAGE (10% polyacrylamide gel), and proteins were visualized by Coomassie Brilliant Blue R-250. Lane M, Marker proteins (the sizes are indicated).
Figure 3.
Figure 3.
pNPX saturation curves of the three purified enzymes. Fractions exhibiting β-d-xylosidase activity obtained after Superdex 200 treatment were pooled (Fig. 1). Aliquots containing approximately 0.1 μg of protein were assayed for β-d-xylosidase activity in a total volume of 0.5 mL. The mixture was incubated at 37°C for 1 h in the presence of increasing concentrations of pNPX from 0.2 to 4.0 mm. The insert presents the corresponding double-reciprocal plot for the determination of Km.
Figure 4.
Figure 4.
Alignment of XYL4, XYL1, and ARAf amino acid sequences. Identical residues in the three sequences are shown in bold. The XYL4, XYL1, and ARAf cDNAs encode polypeptides of 748, 774, and 678 amino acids, respectively. The start of the NH2-terminal residue, after removing signal sequences, is shown by arrowhead. A vertical line is used to indicate the likely COOH terminus, and asterisks indicate potential N-glycosylation sites. Arrows indicate the putative catalytic nucleophiles (Glu-464, Asp-308, and Asp-296 for ARAf, XYL4, and XYL1, respectively) and putative catalytic acid/bases (Glu-388, Glu-512, and Glu-500 for ARAf, XYL4, and XYL1, respectively). Overlines and underlines indicate peptide sequencing with MALDI-TOF after proteolytic cleavage by trypsin for XYL4, XYL1, and ARAf, respectively.
Figure 5.
Figure 5.
Chromatographic analysis of β-d-xylosidase activity at different stages of stem development. A, Elution profile of β-d-xylosidase activity after cation exchange chromatography on CM-Trisacryl at different stages of stem development. A 0.5-mL dialyzed cell-free stem tissue extract (1 mg of protein) was eluted as described in Figure 2. Aliquots of 100 μL were assayed for β-d-xylosidase activity in the presence of 2 mm pNPX. Analysis was performed with stems of 3 to 4 cm and of 11 to 12 cm in length, and with stems at the flowering stage and at the silique stages of development. B, Proportions of the three peaks of β-d-xylosidase activity at the different stages of stem development. Results are expressed as a percentage of the specific activity obtained in comparison with each peak in stems of 3 to 4 cm in length.
Figure 6.
Figure 6.
Phylogenetic trees of plants on the basis of the purified enzymes and homologs. A, For XYL1 and XYL4. B, For ARAf. Branch lengths are drawn to scale. The GenBank accession numbers of the protein sequences are shown. Characterized enzymes are indicated in bold. The BLAST analysis were restricted to proteins from barley, rice (Oryza sativa), and Arabidopsis characterized until December 2003 and showing more than 40% sequence identities with purified enzymes.
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