A beta-glucosidase from lodgepole pine xylem specific for the lignin precursor coniferin.
Coniferin, the glucoside of the monolignol coniferyl alcohol, accumulates to high levels in gymnosperms during spring-cambial reactivation. A cinnamyl alcohol glucoside/beta-glucosidase system is thought to play a key role in lignification by releasing the monolignol aglycones. Investigation of such an enzyme system in the xylem of Pinus contorta var latifolia Engelm. revealed two major beta-glucosidases. One efficiently hydrolyzed the native substrate, coniferin, and the other was more active against synthetic glucosides. The coniferin beta-glucosidase was purified to apparent homogeneity using anion exchange, hydrophobic interaction, and size-exclusion chromatography. The apparent native molecular weight was estimated to be 60,000. A dominant 28-kD protein and a minor 24-kD protein were detected in the purified preparation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunological evidence from polyclonal antibodies directed against the synthetic N-terminal peptide of the 24-kD protein suggested that the native protein is a dimer of 28-kD subunit size. The N-terminal sequence showed that coniferin beta-glucosidase has high homology to known plant beta-glucosidases. Coniferin, syringin, and a synthetic coniferin analog were preferred substrates for the coniferin beta-glucosidase. In situ localization using the chromogenic coniferin analog showed the exclusive presence of beta-glucosidase activity in the differentiating xylem, similar to peroxidase activity.
Dharmawardhana
D P
DP
Biotechnology Laboratory, University of British Columbia, Vancouver, Canada.
Ellis
B E
BE
Carlson
J E
JE
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002934:Cinnamates / Q000378:metabolism*; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006651:Histocytochemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014197:Trees / Q000201:enzymology*; D001617:beta-Glucosidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D002934:Cinnamates; D017886:Durapatite; D001617:beta-Glucosidase; C016316:coniferin