cDNA cloning and heterologous expression of coniferin beta-glucosidase

Coniferin beta-glucosidase (CBG) catalyzes the hydrolysis of monolignol glu cosides to release the cinnamyl alcohols for oxidative polymerization to li gnin. Utilizing the N-terminal amino acid sequence of the purified enzyme, the corresponding full-length cDNA sequence was isolated from a Pinus conto rta 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 h ydrolases, the sequence identity with the N-terminal amino acid residues of the purified enzyme, and the coniferin hydrolytic activity and substrate s pecificity profile displayed by the recombinant protein when expressed in E scherichia 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 prote in targeted to the ER. The isolation of CBG cDNA will facilitate the evalua tion of the importance of this enzyme in the ultimate stages of lignin bios ynthesis and could be a valuable tool in manipulating lignin levels in xyle m cell walls.