|Kuhajek, Jeanne - UNIVERSITY OF MISSISSIPPI|
|Canel, Camilo - NPURU (NOW W/ CORNING)|
|Moraes, Rita - UNIVERSITY OF MISSISSIPPI|
Submitted to: Biochimica et Biophysica Acta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 23, 2002
Publication Date: February 28, 2003
Interpretive Summary: The plant podophyllum peltatum (mayapple) native to the US is known to produce podophyllotoxin (a compound used in the synthesis of an anticancer drug). This plant detoxifies and stores the compound by attaching glucose to it. This paper describe the presence of an enzyme that specifically removes this sugar group (beta glucosidase). This research demonstrated the existence of a substrate specific glucosidase that provided the enhanced yield in podophyllotoxin described in the US patent 6,143,304. A full biochemical characterization of this enzyme is presented. The outcome of this research strengthens the claims made in the patent.
Technical Abstract: A beta-glucosidase with high specificity for podophyllotoxin-4-O-b-d-glucopyranoside was purified from the leaves of Podophyllum peltatum. The 65 kD polypeptide had optimum activity at pH 5.0 and was essentially inactive at physiological pH (6.5 or above). The maximum catalytic activity of this glucosidase was obtained at 45C, but the enzyme was not thermostable and lost 50% of its activity after 18 min at 55C. Inhibition of the enzyme by metal ions Hg2+ or Ag2+ and subsequent reversion of inhibition by b-mercaptoethanol suggest that cysteine residues participate in the catalytic reaction mechanism. This b-glucosidase displayed higher substrate specificity for podophyllotoxin-4-O-b-d-glucopyranoside than epipodophyllotoxin -4-O-b-d- glucopyranoside, and for the (1-3) linkage of laminaribiose than the other glucosidic linkages. The substrate specificity and dependence on a relatively low pH for optimum activity is consistent with a b-glucosidase involved in plant defense mechanisms that would become activated upon cell injury by offending organisms, causing a concomitant lowering of pH and release of lignan glucosides.