- Email: [email protected]
5-hydroxyferulic acid O-methyltransferase from aspen
Phyrochemist~y,VoL 31, No. S, pp. 1495.-1498,1992 Printed in Great Britain.
0031-9422/92$5.00+ 0.00 0 1992Pergamon Press plc
CHARACTERIZATION 5-HYDROXYFERULI~
OF BISPECIFIC CAFFEIC ACID,’ ACID 0-METHYLTRANSFERASE FROM ASPEN
ROBERT C. BUGOS, VINCENT L. C. CHIANG and WILBUR H. CAMPBELL Phytotechnology
Research Center, School of Forestry and Wood Products and Department of Biological Sciences, Michigan Technological University, Houghton, MI 49931, U.S.A. (Received 23 September 1991)
Key Word index--Pop&s tremufoides; Salicaceae; trembling or quaking aspen; developing secondary xylem; bispecific 0-methyltransferase; lignin biosynthesis.
Abstract-Bispecific 0-methyltransferase (OMT, EC 2.1.1.68) which catalyses the meta-specific methylation of caffeic acid and 5-hydroxyferulic acid was purified to homogeneity from the developing secondary xylem of aspen (Populus tremutoides). The enzyme was purified by conventional techniques and affinity chromatography on S-adenosyl-Lhomocysteine agarose using substrate elution. The enzyme has a M, of 40 Ooo as determined by SDS-PAGE. Amino acid sequences of three peptides produced from a proteolytic digest of bispecific OMT were obtained by automated Edman degradation. Polyclonal antibodies raised against the purified OMT reacted strongly to OMT in both,pu~~ed and unpurified fractions in western blots. Addition of an equal concentration of anti-OMT IgG to crude extract protein inhibited OMT activity by more than 70%.
JNTRODUCTION ~-Methy~transferases (OMT) methyIation of 3&dihydroxy
cataiyse a meta-specific cinnamic acids using S-
adenosyI+methionint: (SAM) as the methyl donor [I, 21. Analysis of OMT activity in various species demonstrated that differences in substrate specificity exist between OMTs of gymnosperms (softwoods) and angiosperm dicots (hardwoods) [3-51. Angiosperm dicot OMTs catalyse the methylation of caEeic acid and 5hydroxyferulic acid to form ferulic acid and sinapic acid, respectively. Gymnosperm OMTs catalyse the methylation of &eic acid, but are ine~cient at catalysing the methylation of 5-hydroxyferulic acid. The lack of efficient methyiation of 5-hydroxyferulic acid catalyzed by gymnosperm OMTs suggests one reason for the absence of syringyl lignin in gymnosperms [3-61. Gymnosperm OMTs are therefore considered monospecific, whereas angiosperm dicot OMTs are considered bispecific since they catafyse the methylation of both caffeic acid and 5hydroxyferuIic acid 131. We report the purification to homogeneity of bispecific OMT from the developing secondary xylem of aspen and production of monospecific poIyclona1 antibodies. Purification of a bispecific OMT from aspen (Populus ewamericana) had been attempted earlier [7] but only a t7-fold purification could be obtained by conventional techniques. Anti-OMT antibodies developed in this study have been used to select a cDNA clone encoding bispecific OMT from aspen [S]. RESULTS AND DISCUSSION Aspen bispecific OMT was purified over Ill-fold from the developing secondary xylem of aspen using a four-
step purification protocol including conventional techniques and affinity chromatography (Table 1). The key step in the pu~fication procedure was afYmity chromatography using S-adenosyl-L-homocysteine (SAH) agarose in which SAH was covaiently linked through its u-amino group to acetamidohexyi agarose via a secondary amino linkage. SAH is a competitive inhibitor of this enzyme as
well as other OMTs [S-12]. After applying the partially purified extract to the affinity media and an extensive wash, OMT was eluted with 50gM SAM. With this purified fraction only three protein bands were observed with SDS-PAGE [ 133. This is the first report of a plant OMT to be eluted from this affinity media by the substrate SAM. OMTs from alfalfa root nodules [ 121 and alfalfa suspension cultures [14] were purified on SAHSepharose (SAH attached through its carboxyl group to AH-Sepharose 4B [15]) but elution was performed by decreasing the pH of the buffer. Aspen bispecific OMT was also found to bind to this support but the enzyme would not elute with the addition of SAM to the wash buffer. Purification was also attempted using SAHagarose [SAH covalently linked through its e-amino group to Affi-Gel 10 (Bio-Rad) via an amide linkage] but OMT failed to bind to this support. In a similar type of purification three OMTs from tobacco leaves were puriEed on adenosine-agarose with substrate elution using SAM [16]. Recently, an OMT purified from parsley cell culture, caffeoyl-CoA 3-0-methyltransferas, was partially purified by chromatography on Blue Sepharose CL-6B, but this enzyme would not bind to SAHSepharose or adenosine-agaro~ [ 171. This purifica~on step was attempted with bispecific OMT but the enzyme would not bind to Blue Sepharose. Homogeneous OMT as determined [13] was obtained by chromatography
1495
by SDS-PAGE of the concen-
R. C. Bu~os
1496 Table
1. Purification
Purification step Crude extractt Ammonium sulphate DEAE-Cellulose SAH-Agarose:
of bqeclfic
et al.
OMT from aspen developmg
secondary
xylem
(mg)
Specific activity (pkat mg- ‘)
Purification (-fold)
Activity ratio (5-HFA/CA*)
28.0 7.8 2.6 0.025
156 643 1670 26700
1 4 11 171
2.2 2.1 2.6 3.1
Total protein
*Abbreviations: 5-HFA (5-hydroxyferulic acid); CA (caffeic aad). tExtraction of 15 g developing secondary xylem tissue. IAffinity chromatography using S-adenosyl-L-homocysteine agarose.
trated SAH-agarose eluate on a HPLC gel filtration column at ambient temperature. This purification step is not summarized in the purification table since OMT lost considerable activity at this step. Only one protein peak had activity with caffeic acid and S-hydroxyferulic acid, and this peak consisted of homogeneous protein of M, ca 40 000 as evident by silver staining. Using this purification protocol, OMT was purified to homogeneity and used to raise antibodies. Other OMTs have previously been purified to homogeneity from alfalfa root nodules [ 123, alfalfa suspension cultures [ 141, tobacco leaves [ 16, 181 and parsley cell culture [17]. One of the problems associated with extraction of aspen xylem is browning of the crude extract, even with the addition of polyvinylpolypyrrolidone (PVPP). Browning of crude extracts of xylem from Populus euramericana was attributed to phenoloxidase activity in the extract [7]. Browning of extracts in this study was overcome by addition of 5 mM thiourea, a copper chelator which inhibits phenoloxidase activity, to the extraction buffer [19], Extracts of xylem tissue co!lected late in the growing season had the most problems with browning. The yield of enzyme from extraction was increased by first powdering the tissue in a mortar using liquid nitrogen followed by homogenization in a blender with extraction buffer. This resulted in an almost fourfold increase in the total activity of the enzyme. Substrate specificity of bispecific OMT was tested using a crude extract of aspen developing secondary xylem and five phenylpropanoid substrates (Table 2). The assays contained crude extract (45 pg protein), 0.5 mM SAM and 1 mM phenylpropanoid substrate. Only caffeic acid and 5-hydroxyferulic acid were methylated, indicating that OMT catalyses a meta-specific methylation. pCoumaric acid, ferulic acid and sinapic acid did not serve as substrates, which indicates OMT does not catalyse the methylation of the p-hydroxyl of these substrates. In addition, ferulic acid and sinapic acid, products of metamethylation of caffeic acid and 5-hydroxyferulic acid catalysed by OMT, respectively, do not undergo any further reactions (such as methylation of the p-hydroxyl or methylation of the side chain) that could interfere with the OMT assay. A subunit M, of 40000 was determined for bispecific OMT by SDS-PAGE. Similar subunit M,s were determined for OMTs from alfalfa (41000), pea (38 000) and tobacco (38-43 000) [14,18,20]. However, the number of subunits for these enzymes and aspen bispecific OMT still remains unresolved due to discrepencies in the native M,
Table
2. Substrate
specificity
of aspen bispecific OMT
Substrate*
Relative activity (%)
p-Coumaric acid Caffeic acid Ferulic acid S-Hydroxyferulic acidt Sinapic acid
0 44.0 0 100 0.3
Enzyme assays were perfor’med in duplicate using a crude extract of aspen developing secondary xylem (45 pg protein per assay). *The enzyme assay contained 1 mM phenylpropanoid substrate. tSpeciIIc activity was 432+ 10 pkat 5-hydroxyferulic acid methylated per mg protein.
of these enzymes. Bispecific OMT has a native M, of 60000 as determined by HPLC gel filtration. This M, is 1.5 times the M, of the denatured OMT and does not provide a clear indication of the number of subunits of native OMT. Native M,s of 72000 for aspen OMT [7] and 67000 for Japanese black pine OMT [4] were determined by gel filtration and are very similar to the native M, of bispecific OMT. Subunit M,s were not determined for these enzymes. In addition, OMTs from tobacco [18] and pea [20] had ratios of native to denatured M, of 1.5 which is the same value determined for bispecific OMT. It was suggested that the conformation and size of the OMT proteins apparently affect the gel filtration mobilities of these enzymes [20]. Recently, an OMT from parsley cell culture involved in the formation of cell wall ferulic esters was purified and shown to have a subunit M, of 24000, and a native enzyme composed of two subunits [17,21]. This enzyme not only differs from other plant OMTs in M, but also in substrate specificity. The enzyme methylates caffeoyl-CoA, instead of caffeic acid as catalysed by OMTs of aspen [7], alfalfa [12, 141, pine [4] and tobacco [16, IS]. Peptide sequences of purified bispecific OMT were obtained by automated Edman degradation using standard microsequencing procedures. Amino terminal sequencing of the OMT protein showed it to be blocked, so the purified OMT was digested with Staphylococcus aureus endoproteinase Glu-C and the resulting peptides
Bispecific 0-methyltransferase
of aspen
1497
Table 3. Inhibition of bispecific OMT activity by anti-OMT polyclonal IgG Caffeic acid
Treatment
Residual activity (pkat mg - ‘)
Inhibition (%)
Residual activity (pkat mg- ‘)
Inhibition (%)
Untreated* Preimmune IgGi Anti-OMT IgGt
214_+1 213&O 59*1
_ 1 72
482_+4 470 + 22 138?3
3 71
*Crude extract protein addition of enzyme assay tCrude extract protein 30 pg purified IgG before
(30 pg) was preincubated for 10 min at room temperature before components. (30 pg) was preincubated for 10 min at room temperature with addition of enzyme assay components.
were purified by reversed-phase HPLC. Three internal peptides were sequenced and are listed in standard one
letter amino acid abbreviations: (1) Y-H-G-T-D-P-R-FN-K-V-F-N-K-G-M-S-D-H-S, (2) F-E-G-L-A-K-G-AG-F-Q-G and (3) V-M-C-C-A-F-N-T-H-V-I. Recently, amino acid sequences of four tryptic peptides of caffeoylCoA OMT were obtained [17] and have no significant homology to peptide sequences of bispecific OMT. Polyclonal antibodies were prepared against purified native OMT in a rabbit. Antibodies have previously been prepared against OMTs of tobacco [lS] and parsley [17]. The antibody was purified on recombinant protein A agarose to obtain the IgG fraction, which was used for specificity determination. Protein obtained from each step in the purification of OMT (Table 1 and gel filtration step) were separated by SDS-PAGE and blotted to nitrocellulose. In this western blot, the anti-OMT IgG bound only to a single band of M, 40000at each stage of the purification which establishes the anti-OMT as monospecific. The anti-OMT antibodies reacted strongly to partially purified and purified native OMT when dotted on nitrocellulose, demonstrating that anti-OMT IgG recognizes both native and denatured OMT. AntiOMT IgG also inhibited OMT activity in crude extracts (Table 3). With an equal concentration of crude extract protein and anti-OMT IgG (30 pg each) preincubated for 10 min at room temperature before addition of assay components, OMT activity was inhibited by 70% when assayed with either caffeic acid or 5hydroxyferulic acid. Preimmune IgG had little or no effect on OMT activity. Anti-OMT has since been used to select a lignin specific OMT clone from an aspen developing secondary xylem cDNA library [8] and an OMT clone from an alfalfa cDNA library [22]. EXPERIMENTAL Plant materials.
5-Hydroxyferulic acid
The bark was peeled from 3-4-year-old aspen trees (Pop&s tremuloides) growing on our campus, exposing the developing secondary xylem on the woody stem. This layer (0.2-0.5 mm) was scraped from the stem with a razor blade and immediately frozen in liquid N,. Developing secondary xylem was collected during the months of June through August. &firs. Buffer A was 200 mM KPi (pH 7.7) containing 5 mM NaN,, 1 mM EDTA, 5 mM 2-mercaptoethanol and 5 mM thourea. Buffer B was 20 mM KPi (pH 7.7) containing 5 mM NaN,, 1 mM EDTA and 5 mM 2-mercaptoethanol. Buffer C
was 5 mM KPi (pH 7.7) containing 1 mM EDTA and 5 mM 2-mercaptoethanol. Extraction and purijication of OMTfrom aspen. Developing secondary xylem tissue was pulverized in a mortar in the presence of liquid N, and homogenized in a blender with buffer A (4 ml g-’ tissue) containing PVPP (0.1 gg-’ tissue) for 30-60 sec. The homogenate was filtered through 2 layers of Miracloth (Calbiochem) and centrifuged at 10000 g for 20 min. Ail purification steps were performed at 4” unless otherwise stated. The supernatant was treated with solid (NH&SO,, centrifuged and the protein that pptd between 30&O% satn was dissolved in a minimal vol. of buffer B, and dialysed against 3 1 of the same buffer overnight. The dialysed extract was centrifuged at 10000 g for 10min to remove pptd protein, and the supernatant was applied to a DEAE-cellulose column previously equilibrated with buffer B. The column was washed with buffer B until no A was detected at 280 nm and eluted with buffer B containing 150 mM KCl. Fractions containing the highest OMT activity were pooled and dialysed overnight against 3 1 of buffer C. The dialysed DEAEcellulose eluate was applied to an SAH-agarose column (BRL) previously equilibrated with buffer C. After an extensive wash of the column with this buffer, the enzyme was eluted with 50 PM SAM (Boehringer) in buffer C. Fractions with OMT activity were pooled and the soln was coned to ca 1 ml with a Centriconmicroconcentrator (Amicon). The coned soln was chromatographed on a TSK-Gel 3000SW column (Phenomenex, 7.5 mm i.d. x 50 cm) equilibrated with 100 mM NaPi (PH 6.9) at a flow rate of 1 ml min- ’ at ambient temp. The protein peaks monitored at 214 nm were collected at the detector and unmediately placed on ice. The fraction with enzyme activity (retention time 15.3 min) was coned to ca 200 ~1 with a Centricon-30 microconcentrator and stored at 4”. Protein determination. Protein content was determined with a prepared dye binding reagent (Bio-Rad) using BSA as the standard. A micro-protocol adapted for photometry with a microtitre plate reader was used with low cones of protein [23]. OM T assay. The OMT activity assay was based on a previous protocol [7]. All assays were performed in duplicate. The reaction mixture (200 pl) contained 50 mM Tris (pH 8), 10 mM MgCI,, 1 mM phenylpropanoid substrate (caffeic or 5-hydroxyferulic acid) and l-100 ~1enzyme soln in a 1.5 ml microfuge tube. 5-Hydroxyferuhc acid was synthesized by condensing 3methoxy-4,5-dihydroxybenzaldehyde (Pfaltz & Bauer) and malonic acid 1241.After preincubation for 5 min at 30”, 100 nmol of diluted SAM-“‘Me (1 ~1) was added and the reaction was incubated for 10 min at 30”. The diluted SAM-14Me was prepared by adding 4.9 mg SAM .HSO, (Bochringcr) to 100 pl
1498
R. C. BUG~S et al.
SAM-14Me (54 mC1 mmol _ ‘) (Amersham). The reaction was terminated by the addition of 20 ~1 of 2 M HCl. Et,0 (1 ml) was added and the contents were shaken/vortexed vigorously and centrifuged briefly to separate the aq. and Et,0 phases. The reaction mixture was placed at - 70” for 30 min to freeze the aq. phase and the Et,0 was transferred to a scintillation vlal. Radioactivity was determined by liquid scintillation after addition of S ml Bio-Safe II counting cocktail (Research Products International). Control assays contained no phenylpropanoid substrate. Polyacrylamide gel electrophoresls. Proteins were separated under denaturing conditions using SDS-PAGE. Acrylamide slab gels were prepared essentially as described [25] with 4% stackmg gels and 10% resolving gels. Samples were boiled in treatment buffer and electrophoresed usmg a mini-gel electrophoresis unit according to the manufacturer’s instructions (Hoefer Screnhfic). Following electrophoresis, proteins were stamed using a silver staming kit (Bio-Rad). Antibody production and IgG purijication. Purified OMT (70 pg) was emulsified with an equal vol. of Freund’s complete adJuvant and inJected into multiple subcutaneous sites on the back of a New Zealand white rabbit. The injection was repeated 3 weeks later with 36 /Ig OMT emulsified with an equal vol. of Freund’s incomplete adjuvant. After one week, an ear bleed was performed and the resulting serum was found to inhibit OMT activity. Serum was collected from the rabbit and stored at -20”. The IgG fraction was purified by chromatography on recombinant Protein A agarose (Repligen) and quantitated as described C261. Western blottmg. Proteins were transferred to nitrocellulose (S & S) essentially as previously described [27]. Transfer was performed at 50 V for 3 hr at 4”. The blot was developed by immunodetection essentially as described [28]. After blocking non-specific protein binding sites with 3% BSA for 1 hr, the blot was Incubated for 2 hr with 1 pgml-’ anti-OMT IgG. Alkaline phosphatase conjugated goat anti-rabbit IgG (Cappel) was incubated with the blot for 45 mm, followed by colour development with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. Protem sequencing. Proteolytic digestion of blspectific OMT with Staphylococcus aureus endoproteinase Glu-C, purification of peptldes by reversed-phase HPLC and automated Edman degradation were performed at the University of Wisconsin Biotechnology Center. Acknowledgements--This research was supported by United States Department of Agriculture Competitive Research Grant 90-37291-5708 of the Forest Biology Program.
REFERENCES 1. Grisebach, H. (1981) in The Biochemistry of Plants Vol. 7 (Stumpf, P. K. and Corm, E. E., eds), p. 457. Academic Press, New York.
2. Gross, G. G. (1985) in Biosynthesis and Biodegradation of Wood Components (Higuchi, T., ed.), p. 229. Academic Press, Orlando. 3. Shimada, M., Fushlki, H. and Higuchi, T. (1973) Mokuzai Gakkaishi 19, 13. 4. Kuroda, H., Shimada, M. and Higuchi, T. (1975) Phytochemistry 14, 1759. F. and Tanahashi, M. 5. Higuchi, T., Shimada, M., Nakatsubo. (1977) Wood Sci. Technol. 11, 153. 6. Shimada, M.. Fushiki, H. and Higuchi, T. (1972) Phytochemistry 11, 2657 7. Kuroda, H., Shimada. M. and Higuchi, T. (1981) Phytochemistry 20, 2635. 8. Bugos, R. C., Chiang, V. L. and Campbell, W. H. (1991) Plant Mol. &al. 17. 1203. 9. Collendavelloo, J., Legrand, M., Geoffroy, P., Barthelemy, J. and Fritig, B. (1981) Phytochemistry 20, 611. 10. Poulton, J. E. and Butt. V. S. (1975) E&him. Biophys. Acta 403, 301. 11. Poulton, J., Hahlbrock, K. and Grisebach, H. (1976) Arch. Biochem. Biophys. 176, 449. 12. Vance, C. P. and Bryan, J. W. (1981) Phytochemistry 20,41. 13. Bugos, R. C., Chiang V. L. and Campbell, W. H. (1989) in TAPPI Proceedings, Wood and Pulping Chemistry, p. 345. TAPPI Press, Atlanta. 14. Edwards, R. and Dixon, R. A. (1991) Arch. Biochem. Biophys. 287, 372. 15. Sharma, S. K. and Brown, S. A. (1978) J. Chromatogr. 157, 247. 16. Dumas, B., Legrand, M., Geoffroy. P. and Fritig, B. (1988) Planta 176, 36. 17. Pakusch, A. E., Matern, U. and Schiltz, E. (1991) Plant Physiol. 95, 137. 18. Hermann, C., Legrand, M., Geoffroy, P. and Fritig, B. (1987) Arch. Biochem. Biophys. 253, 367. 19. Van Driessche, E., Beeckmans, S., DeJaegere, R. and Kanarck, L. (1984) Analyt. Biochem. 141, 184. 20. Preisig, C. L., Matthews, D. E. and VanEtten, H. D. (1989) Plant Physiol. 91, 559. 21. Pakusch, A. E., Kneusel, R. E. and Matern, U. (1989) Arch. Biochem. Biophys. 271, 488. 22. Gown, G., Bugos, R. C., Campbell, W. H., Maxwell, C. A. and Dixon, R. A. (1991) Pfant Physiol. 97, 7. 23. Redinbaugh, M. G. and Campbell, W. H. (1985) Analyt. Biochem. 147, 144. 24. Neish, A. C. (1959) Can. J. Blochem. Physiol. 37, 1431. 25. Laemmli, U. K. (1970) Nature 227, 680. 26. Hyde, G. E., Wilberding, J. A., Meyer, A. L., Campbell, E. R. and Campbell, W. H. (1989) Plant Mol. Biol. 13, 233. 27. Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natn. Acad. Sci. U.S.A. 76. 4350. 28. Mierendorf, R. C., Percy, C. and Young, R. A. (1987) Meth. Enzymol. 152,458.
0031-9422/92$5.00+ 0.00 0 1992Pergamon Press plc
CHARACTERIZATION 5-HYDROXYFERULI~
OF BISPECIFIC CAFFEIC ACID,’ ACID 0-METHYLTRANSFERASE FROM ASPEN
ROBERT C. BUGOS, VINCENT L. C. CHIANG and WILBUR H. CAMPBELL Phytotechnology
Research Center, School of Forestry and Wood Products and Department of Biological Sciences, Michigan Technological University, Houghton, MI 49931, U.S.A. (Received 23 September 1991)
Key Word index--Pop&s tremufoides; Salicaceae; trembling or quaking aspen; developing secondary xylem; bispecific 0-methyltransferase; lignin biosynthesis.
Abstract-Bispecific 0-methyltransferase (OMT, EC 2.1.1.68) which catalyses the meta-specific methylation of caffeic acid and 5-hydroxyferulic acid was purified to homogeneity from the developing secondary xylem of aspen (Populus tremutoides). The enzyme was purified by conventional techniques and affinity chromatography on S-adenosyl-Lhomocysteine agarose using substrate elution. The enzyme has a M, of 40 Ooo as determined by SDS-PAGE. Amino acid sequences of three peptides produced from a proteolytic digest of bispecific OMT were obtained by automated Edman degradation. Polyclonal antibodies raised against the purified OMT reacted strongly to OMT in both,pu~~ed and unpurified fractions in western blots. Addition of an equal concentration of anti-OMT IgG to crude extract protein inhibited OMT activity by more than 70%.
JNTRODUCTION ~-Methy~transferases (OMT) methyIation of 3&dihydroxy
cataiyse a meta-specific cinnamic acids using S-
adenosyI+methionint: (SAM) as the methyl donor [I, 21. Analysis of OMT activity in various species demonstrated that differences in substrate specificity exist between OMTs of gymnosperms (softwoods) and angiosperm dicots (hardwoods) [3-51. Angiosperm dicot OMTs catalyse the methylation of caEeic acid and 5hydroxyferulic acid to form ferulic acid and sinapic acid, respectively. Gymnosperm OMTs catalyse the methylation of &eic acid, but are ine~cient at catalysing the methylation of 5-hydroxyferulic acid. The lack of efficient methyiation of 5-hydroxyferulic acid catalyzed by gymnosperm OMTs suggests one reason for the absence of syringyl lignin in gymnosperms [3-61. Gymnosperm OMTs are therefore considered monospecific, whereas angiosperm dicot OMTs are considered bispecific since they catafyse the methylation of both caffeic acid and 5hydroxyferuIic acid 131. We report the purification to homogeneity of bispecific OMT from the developing secondary xylem of aspen and production of monospecific poIyclona1 antibodies. Purification of a bispecific OMT from aspen (Populus ewamericana) had been attempted earlier [7] but only a t7-fold purification could be obtained by conventional techniques. Anti-OMT antibodies developed in this study have been used to select a cDNA clone encoding bispecific OMT from aspen [S]. RESULTS AND DISCUSSION Aspen bispecific OMT was purified over Ill-fold from the developing secondary xylem of aspen using a four-
step purification protocol including conventional techniques and affinity chromatography (Table 1). The key step in the pu~fication procedure was afYmity chromatography using S-adenosyl-L-homocysteine (SAH) agarose in which SAH was covaiently linked through its u-amino group to acetamidohexyi agarose via a secondary amino linkage. SAH is a competitive inhibitor of this enzyme as
well as other OMTs [S-12]. After applying the partially purified extract to the affinity media and an extensive wash, OMT was eluted with 50gM SAM. With this purified fraction only three protein bands were observed with SDS-PAGE [ 133. This is the first report of a plant OMT to be eluted from this affinity media by the substrate SAM. OMTs from alfalfa root nodules [ 121 and alfalfa suspension cultures [14] were purified on SAHSepharose (SAH attached through its carboxyl group to AH-Sepharose 4B [15]) but elution was performed by decreasing the pH of the buffer. Aspen bispecific OMT was also found to bind to this support but the enzyme would not elute with the addition of SAM to the wash buffer. Purification was also attempted using SAHagarose [SAH covalently linked through its e-amino group to Affi-Gel 10 (Bio-Rad) via an amide linkage] but OMT failed to bind to this support. In a similar type of purification three OMTs from tobacco leaves were puriEed on adenosine-agarose with substrate elution using SAM [16]. Recently, an OMT purified from parsley cell culture, caffeoyl-CoA 3-0-methyltransferas, was partially purified by chromatography on Blue Sepharose CL-6B, but this enzyme would not bind to SAHSepharose or adenosine-agaro~ [ 171. This purifica~on step was attempted with bispecific OMT but the enzyme would not bind to Blue Sepharose. Homogeneous OMT as determined [13] was obtained by chromatography
1495
by SDS-PAGE of the concen-
R. C. Bu~os
1496 Table
1. Purification
Purification step Crude extractt Ammonium sulphate DEAE-Cellulose SAH-Agarose:
of bqeclfic
et al.
OMT from aspen developmg
secondary
xylem
(mg)
Specific activity (pkat mg- ‘)
Purification (-fold)
Activity ratio (5-HFA/CA*)
28.0 7.8 2.6 0.025
156 643 1670 26700
1 4 11 171
2.2 2.1 2.6 3.1
Total protein
*Abbreviations: 5-HFA (5-hydroxyferulic acid); CA (caffeic aad). tExtraction of 15 g developing secondary xylem tissue. IAffinity chromatography using S-adenosyl-L-homocysteine agarose.
trated SAH-agarose eluate on a HPLC gel filtration column at ambient temperature. This purification step is not summarized in the purification table since OMT lost considerable activity at this step. Only one protein peak had activity with caffeic acid and S-hydroxyferulic acid, and this peak consisted of homogeneous protein of M, ca 40 000 as evident by silver staining. Using this purification protocol, OMT was purified to homogeneity and used to raise antibodies. Other OMTs have previously been purified to homogeneity from alfalfa root nodules [ 123, alfalfa suspension cultures [ 141, tobacco leaves [ 16, 181 and parsley cell culture [17]. One of the problems associated with extraction of aspen xylem is browning of the crude extract, even with the addition of polyvinylpolypyrrolidone (PVPP). Browning of crude extracts of xylem from Populus euramericana was attributed to phenoloxidase activity in the extract [7]. Browning of extracts in this study was overcome by addition of 5 mM thiourea, a copper chelator which inhibits phenoloxidase activity, to the extraction buffer [19], Extracts of xylem tissue co!lected late in the growing season had the most problems with browning. The yield of enzyme from extraction was increased by first powdering the tissue in a mortar using liquid nitrogen followed by homogenization in a blender with extraction buffer. This resulted in an almost fourfold increase in the total activity of the enzyme. Substrate specificity of bispecific OMT was tested using a crude extract of aspen developing secondary xylem and five phenylpropanoid substrates (Table 2). The assays contained crude extract (45 pg protein), 0.5 mM SAM and 1 mM phenylpropanoid substrate. Only caffeic acid and 5-hydroxyferulic acid were methylated, indicating that OMT catalyses a meta-specific methylation. pCoumaric acid, ferulic acid and sinapic acid did not serve as substrates, which indicates OMT does not catalyse the methylation of the p-hydroxyl of these substrates. In addition, ferulic acid and sinapic acid, products of metamethylation of caffeic acid and 5-hydroxyferulic acid catalysed by OMT, respectively, do not undergo any further reactions (such as methylation of the p-hydroxyl or methylation of the side chain) that could interfere with the OMT assay. A subunit M, of 40000 was determined for bispecific OMT by SDS-PAGE. Similar subunit M,s were determined for OMTs from alfalfa (41000), pea (38 000) and tobacco (38-43 000) [14,18,20]. However, the number of subunits for these enzymes and aspen bispecific OMT still remains unresolved due to discrepencies in the native M,
Table
2. Substrate
specificity
of aspen bispecific OMT
Substrate*
Relative activity (%)
p-Coumaric acid Caffeic acid Ferulic acid S-Hydroxyferulic acidt Sinapic acid
0 44.0 0 100 0.3
Enzyme assays were perfor’med in duplicate using a crude extract of aspen developing secondary xylem (45 pg protein per assay). *The enzyme assay contained 1 mM phenylpropanoid substrate. tSpeciIIc activity was 432+ 10 pkat 5-hydroxyferulic acid methylated per mg protein.
of these enzymes. Bispecific OMT has a native M, of 60000 as determined by HPLC gel filtration. This M, is 1.5 times the M, of the denatured OMT and does not provide a clear indication of the number of subunits of native OMT. Native M,s of 72000 for aspen OMT [7] and 67000 for Japanese black pine OMT [4] were determined by gel filtration and are very similar to the native M, of bispecific OMT. Subunit M,s were not determined for these enzymes. In addition, OMTs from tobacco [18] and pea [20] had ratios of native to denatured M, of 1.5 which is the same value determined for bispecific OMT. It was suggested that the conformation and size of the OMT proteins apparently affect the gel filtration mobilities of these enzymes [20]. Recently, an OMT from parsley cell culture involved in the formation of cell wall ferulic esters was purified and shown to have a subunit M, of 24000, and a native enzyme composed of two subunits [17,21]. This enzyme not only differs from other plant OMTs in M, but also in substrate specificity. The enzyme methylates caffeoyl-CoA, instead of caffeic acid as catalysed by OMTs of aspen [7], alfalfa [12, 141, pine [4] and tobacco [16, IS]. Peptide sequences of purified bispecific OMT were obtained by automated Edman degradation using standard microsequencing procedures. Amino terminal sequencing of the OMT protein showed it to be blocked, so the purified OMT was digested with Staphylococcus aureus endoproteinase Glu-C and the resulting peptides
Bispecific 0-methyltransferase
of aspen
1497
Table 3. Inhibition of bispecific OMT activity by anti-OMT polyclonal IgG Caffeic acid
Treatment
Residual activity (pkat mg - ‘)
Inhibition (%)
Residual activity (pkat mg- ‘)
Inhibition (%)
Untreated* Preimmune IgGi Anti-OMT IgGt
214_+1 213&O 59*1
_ 1 72
482_+4 470 + 22 138?3
3 71
*Crude extract protein addition of enzyme assay tCrude extract protein 30 pg purified IgG before
(30 pg) was preincubated for 10 min at room temperature before components. (30 pg) was preincubated for 10 min at room temperature with addition of enzyme assay components.
were purified by reversed-phase HPLC. Three internal peptides were sequenced and are listed in standard one
letter amino acid abbreviations: (1) Y-H-G-T-D-P-R-FN-K-V-F-N-K-G-M-S-D-H-S, (2) F-E-G-L-A-K-G-AG-F-Q-G and (3) V-M-C-C-A-F-N-T-H-V-I. Recently, amino acid sequences of four tryptic peptides of caffeoylCoA OMT were obtained [17] and have no significant homology to peptide sequences of bispecific OMT. Polyclonal antibodies were prepared against purified native OMT in a rabbit. Antibodies have previously been prepared against OMTs of tobacco [lS] and parsley [17]. The antibody was purified on recombinant protein A agarose to obtain the IgG fraction, which was used for specificity determination. Protein obtained from each step in the purification of OMT (Table 1 and gel filtration step) were separated by SDS-PAGE and blotted to nitrocellulose. In this western blot, the anti-OMT IgG bound only to a single band of M, 40000at each stage of the purification which establishes the anti-OMT as monospecific. The anti-OMT antibodies reacted strongly to partially purified and purified native OMT when dotted on nitrocellulose, demonstrating that anti-OMT IgG recognizes both native and denatured OMT. AntiOMT IgG also inhibited OMT activity in crude extracts (Table 3). With an equal concentration of crude extract protein and anti-OMT IgG (30 pg each) preincubated for 10 min at room temperature before addition of assay components, OMT activity was inhibited by 70% when assayed with either caffeic acid or 5hydroxyferulic acid. Preimmune IgG had little or no effect on OMT activity. Anti-OMT has since been used to select a lignin specific OMT clone from an aspen developing secondary xylem cDNA library [8] and an OMT clone from an alfalfa cDNA library [22]. EXPERIMENTAL Plant materials.
5-Hydroxyferulic acid
The bark was peeled from 3-4-year-old aspen trees (Pop&s tremuloides) growing on our campus, exposing the developing secondary xylem on the woody stem. This layer (0.2-0.5 mm) was scraped from the stem with a razor blade and immediately frozen in liquid N,. Developing secondary xylem was collected during the months of June through August. &firs. Buffer A was 200 mM KPi (pH 7.7) containing 5 mM NaN,, 1 mM EDTA, 5 mM 2-mercaptoethanol and 5 mM thourea. Buffer B was 20 mM KPi (pH 7.7) containing 5 mM NaN,, 1 mM EDTA and 5 mM 2-mercaptoethanol. Buffer C
was 5 mM KPi (pH 7.7) containing 1 mM EDTA and 5 mM 2-mercaptoethanol. Extraction and purijication of OMTfrom aspen. Developing secondary xylem tissue was pulverized in a mortar in the presence of liquid N, and homogenized in a blender with buffer A (4 ml g-’ tissue) containing PVPP (0.1 gg-’ tissue) for 30-60 sec. The homogenate was filtered through 2 layers of Miracloth (Calbiochem) and centrifuged at 10000 g for 20 min. Ail purification steps were performed at 4” unless otherwise stated. The supernatant was treated with solid (NH&SO,, centrifuged and the protein that pptd between 30&O% satn was dissolved in a minimal vol. of buffer B, and dialysed against 3 1 of the same buffer overnight. The dialysed extract was centrifuged at 10000 g for 10min to remove pptd protein, and the supernatant was applied to a DEAE-cellulose column previously equilibrated with buffer B. The column was washed with buffer B until no A was detected at 280 nm and eluted with buffer B containing 150 mM KCl. Fractions containing the highest OMT activity were pooled and dialysed overnight against 3 1 of buffer C. The dialysed DEAEcellulose eluate was applied to an SAH-agarose column (BRL) previously equilibrated with buffer C. After an extensive wash of the column with this buffer, the enzyme was eluted with 50 PM SAM (Boehringer) in buffer C. Fractions with OMT activity were pooled and the soln was coned to ca 1 ml with a Centriconmicroconcentrator (Amicon). The coned soln was chromatographed on a TSK-Gel 3000SW column (Phenomenex, 7.5 mm i.d. x 50 cm) equilibrated with 100 mM NaPi (PH 6.9) at a flow rate of 1 ml min- ’ at ambient temp. The protein peaks monitored at 214 nm were collected at the detector and unmediately placed on ice. The fraction with enzyme activity (retention time 15.3 min) was coned to ca 200 ~1 with a Centricon-30 microconcentrator and stored at 4”. Protein determination. Protein content was determined with a prepared dye binding reagent (Bio-Rad) using BSA as the standard. A micro-protocol adapted for photometry with a microtitre plate reader was used with low cones of protein [23]. OM T assay. The OMT activity assay was based on a previous protocol [7]. All assays were performed in duplicate. The reaction mixture (200 pl) contained 50 mM Tris (pH 8), 10 mM MgCI,, 1 mM phenylpropanoid substrate (caffeic or 5-hydroxyferulic acid) and l-100 ~1enzyme soln in a 1.5 ml microfuge tube. 5-Hydroxyferuhc acid was synthesized by condensing 3methoxy-4,5-dihydroxybenzaldehyde (Pfaltz & Bauer) and malonic acid 1241.After preincubation for 5 min at 30”, 100 nmol of diluted SAM-“‘Me (1 ~1) was added and the reaction was incubated for 10 min at 30”. The diluted SAM-14Me was prepared by adding 4.9 mg SAM .HSO, (Bochringcr) to 100 pl
1498
R. C. BUG~S et al.
SAM-14Me (54 mC1 mmol _ ‘) (Amersham). The reaction was terminated by the addition of 20 ~1 of 2 M HCl. Et,0 (1 ml) was added and the contents were shaken/vortexed vigorously and centrifuged briefly to separate the aq. and Et,0 phases. The reaction mixture was placed at - 70” for 30 min to freeze the aq. phase and the Et,0 was transferred to a scintillation vlal. Radioactivity was determined by liquid scintillation after addition of S ml Bio-Safe II counting cocktail (Research Products International). Control assays contained no phenylpropanoid substrate. Polyacrylamide gel electrophoresls. Proteins were separated under denaturing conditions using SDS-PAGE. Acrylamide slab gels were prepared essentially as described [25] with 4% stackmg gels and 10% resolving gels. Samples were boiled in treatment buffer and electrophoresed usmg a mini-gel electrophoresis unit according to the manufacturer’s instructions (Hoefer Screnhfic). Following electrophoresis, proteins were stamed using a silver staming kit (Bio-Rad). Antibody production and IgG purijication. Purified OMT (70 pg) was emulsified with an equal vol. of Freund’s complete adJuvant and inJected into multiple subcutaneous sites on the back of a New Zealand white rabbit. The injection was repeated 3 weeks later with 36 /Ig OMT emulsified with an equal vol. of Freund’s incomplete adjuvant. After one week, an ear bleed was performed and the resulting serum was found to inhibit OMT activity. Serum was collected from the rabbit and stored at -20”. The IgG fraction was purified by chromatography on recombinant Protein A agarose (Repligen) and quantitated as described C261. Western blottmg. Proteins were transferred to nitrocellulose (S & S) essentially as previously described [27]. Transfer was performed at 50 V for 3 hr at 4”. The blot was developed by immunodetection essentially as described [28]. After blocking non-specific protein binding sites with 3% BSA for 1 hr, the blot was Incubated for 2 hr with 1 pgml-’ anti-OMT IgG. Alkaline phosphatase conjugated goat anti-rabbit IgG (Cappel) was incubated with the blot for 45 mm, followed by colour development with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. Protem sequencing. Proteolytic digestion of blspectific OMT with Staphylococcus aureus endoproteinase Glu-C, purification of peptldes by reversed-phase HPLC and automated Edman degradation were performed at the University of Wisconsin Biotechnology Center. Acknowledgements--This research was supported by United States Department of Agriculture Competitive Research Grant 90-37291-5708 of the Forest Biology Program.
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