Abstract
Coniferin β-glucosidase (CBG) catalyzes the hydrolysis of monolignol glucosides to release the cinnamyl alcohols for oxidative polymerization to lignin. Utilizing the N-terminal amino acid sequence of the purified enzyme, the corresponding full-length cDNA sequence was isolated from a Pinus contorta xylem-specific library. The isolated 1909 nucleotide cDNA was confirmed to be that of CBG on the basis of its high homology to family 1 glycosyl hydrolases, the sequence identity with the N-terminal amino acid residues of the purified enzyme, and the coniferin hydrolytic activity and substrate specificity profile displayed by the recombinant protein when expressed in Escherichia coli. The presence of a 23 amino acid N-terminal signal peptide in the deduced 513 amino acid enzyme suggests that CBG is a secretory protein targeted to the ER. The isolation of CBG cDNA will facilitate the evaluation of the importance of this enzyme in the ultimate stages of lignin biosynthesis and could be a valuable tool in manipulating lignin levels in xylem cell walls.
Similar content being viewed by others
References
Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403–410.
Baird, S.D., Hefford, M.A., Johnson, D.A., Sung, W.L., Yaguchi, M. and Seligy, V.L. 1990. The Glu residue in the conserved Asn-Glu-Pro sequence of two highly divergent endo β-1,4-glucanases is essential for enzyme activity. Biochem. Biophys. Res. Comm. 169: 1035–1039.
Brzobohaty, B., Moore, I. and Kristoffersen, P. 1993. Release of active cytokinin by β-glucosidases localized to the maize root meristem. Science 262: 1051.
Campbell, M.M. and Ellis, B.E. 1992. Fungal elicitor-mediated responses in pine cell cultures. I. Induction of phenylpropanoid metabolism. Planta 180: 409–417.
Castle, L.A., Smith, K.D. and Morris, R.D. 1992. Cloning and sequencing of an Agrobacterium tumefaciens β-glucosidase gene involved in modifying a vir-inducing plant signal molecule. J. Bact. 174: 1478–1486.
Dharmawardhana, D.P., Ellis, B.E. and Carlson, J.E. 1995. A β-glucosidase from lodgepole pine xylem specific for the lignin precursor coniferin. Plant Physiol. 107: 331–339.
Falk, A., Xue, J., Lenman, M. and Rask, L. 1992. Sequence of a cDNA clone encoding the enzyme myrosinase and expression of myrosinase in different tissues of Brassica napus. Plant Sci. 83: 181–186.
Freudenberg, K. and Harkin, J.M. 1963. The glucosides of cambial sap of spruce. Phytochemistry 2: 189–193.
Fukushima, K. and Terashima, N. 1990. Heterogeneity in formation of lignin XIII. J. Wood Chem. Technol. 10: 413–433.
Fukushima, K. and Terashima, N. 1991. Heterogeneity in formation of lignin XIV. Holzforschung 45: 87–89.
Henrissat, B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280: 309–316.
Holton, T.A. and Graham. 1991. A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. Nucl. Acids Res. 191: 1156–1158.
Hrazdina, G. and Jensen, R.A. 1992. Spatial organization of enzymes in plant metabolic pathways. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43: 241–267.
Hrazdina, G. and Wagner, G.J. 1985. Metabolic pathways as enzyme complexes: evidence for the synthesis of phenylpropanoids and flavonoids on membrane-associated enzyme complexes. Arch. Biochem. Biophys. 237: 88–100.
Hughes, M.A. and Dunn, M.A. 1982. Biochemical characterization of the li locus, which controls the activity of the cyanogenic β-glucosidase in Trifolium repens. Plant Mol. Biol. 1: 169–181.
Joshi, C.P. 1987. Putative polyadenylation signals in nuclear genes of higher plants: a compilation and analysis. Nucl. Acids Res. 15: 9627–9640.
Klein, P., Kanehisa, M. and Delisi, C. 1985. Description of one of the methods used in SOAP. Biochim. Biophys. Acta. 815: 468–476.
Lawton, M.A., Clouse, S.D. and Lamb, C.J. 1990. Glutathioneelicited changes in chromatin structure within the promoter of the defense gene chalcone synthase. Plant Cell Rep. 8: 561–564.
Leah, R., Kigel, J., Svendsen, I.B. and Mundy, J. 1995. Biochemical and molecular characterization of barley seed β-glucosidase. J. Biol. Chem.270: 15789–15796.
Leinhos, V. and Savidge, R.A. 1993. Isolation of protoplasts from developing xylem of Pinus banksiana and Pinus strobus. Can. J. For. Res. 23: 343–348.
Leinhos, V., Udagama Randeniya, P.V. and Savidge, R. 1994. Purifi-cation of an acidic coniferin-hydrolysing beta-glucosidase from developing xylem of Pinus banksiana. Phytochemistry (Oxford) 37: 311–315.
Lewinsohn, E., Steele, C. and Croteau, R. 1994. Simple isolation of functional RNA from woody stems of gymnosperms. Plant Mol. Biol. Rep. 12: 20–25.
Ohl, S., Hendrick, S.A., Chory, J. and Lamb, C.J. 1990. Functional properties of a phenylalanine ammonia-lyase promoter from Arabidopsis. Plant Cell 2: 837–848.
Oxtoby, E., Dunn, A., Pancoro, A. and Hughes, M.A. 1991. Nucleotide sequence and derived amino acid sequence of the cyanogenic β-glucosidase (linamarase) from white clover. Plant Mol. Biol. 17: 209–219.
Pickett-Heaps, J.D. 1968. Xylem wall deposition. Radioautographic investigation using lignin precursors. Protoplasma 65: 181–190.
Savidge, R.A. 1988. A biochemical indicator of commitment to tracheid differentiation in Pinus contorta. Can. J. Bot. 66: 2099–2112.
Sinnott, M. 1990. Catalytic mechanisms of glycosyl transfer. Chem. Rev. 90: 1171–1202.
Terashima, N. and Fukushima, K. 1998. Heterogeneity in formation of lignin-XI. Wood Sci. Technol. 22: 259–270.
Terashima, T., Fukushima, K. and Tsuchiya, S. 1986. Heterogeneity in formation of lignin. VII. J. Wood Sci. Technol. 6: 495–504.
Terazawa, M., Okuyama, H. and Miyake, M. 1984. Phenolic compounds in living tissue of woods. I. Phenolic glucosides of 4-hydroxycinnamyl alcohol derivatives in the cambial sap of woods. Mokuzai Gakkaishi 30: 322–328.
Trimbur, D.E., Warren, R.A.J. and Withers, S.G. 1992. Regiondirected mutagenesis of residues surrounding the active site nucleophile in β-glucosidase from Agrobacterium faecalis. J. Biol. Chem. 267: 10248–10251.
Varghese, J.N., Garrett, T.P.J., Colman, P.M., Chen, L., Híj, P.B. and Fincher, G.G. 1994. Three-dimensional structures of two plant β-glucan endohydrolases with distinct substrate specificities. Proc. Natl. Acad. Sci. USA 91: 2785–2789.
von Heijne, G. 1986. A new method for predicting signal sequence cleavage sites. Nucl. Acids Res. 14: 4683–4690.
Whetten, R. and Sederoff, R. 1995. Lignin biosynthesis. Plant Cell 7: 1001–1013.
Withers, S.G., Warren, R.A.J., Street, I.P., Rupitz, J.B., Kempton, J.B. and Aebersold, R. 1990. Unequivocal demonstration of the involvement of a glutamate residue as a nucleophile in the mechanism of a 'retaining' glycosidase. J. Am. Chem. Soc. 112: 5887–5889.
Wu, L., Uede, T. and Messing, J. 1995. The formation of mRNA 30 ends in plants. Plant J. 8: 323–329.
Xue, J., Lenman, M., Falk, A. and Rask, L. 1992. The glucosinolatedegrading enzyme myrosinase in Brassicaceae is encoded by a gene family. Plant Mol. Biol. 18: 387–398.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Dharmawardhana, D.P., Ellis, B.E. & Carlson, J.E. cDNA cloning and heterologous expression of coniferin β-glucosidase. Plant Mol Biol 40, 365–372 (1999). https://doi.org/10.1023/A:1006226931512
Issue Date:
DOI: https://doi.org/10.1023/A:1006226931512