אסיף מאגר המחקר החקלאי

UDP-rhamnose:flavanone-7-O-glucoside-2″-O-rhamnosyltransferase: Purification and characterization of an enzyme catalyzing the production of bitter compounds in citrus

Year:

1991

Source of publication :

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Volume :

266

Co-Authors:

Bar-Peled, M., Department of Plant Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
Lewinsohn, E., Department of Plant Genetics, Weizmann Institute of Science, Rehovot 76100, Israel, Inst. of Biological Chemistry, Pullman, WA 99164, United States
Fluhr, R., Department of Plant Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
Gressel, J., Department of Plant Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

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Abstract:

The rhamnosyltransferase catalyzing the production of the bitter flavanone-glucosides, naringin and neohesperidin, was purified to homogeneity. The enzyme catalyzes the transfer of rhamnose from UDP-rhamnose to the C-2 hydroxyl group of glucose attached via C-7-O- of naringenin or hesperetin. To our knowledge this is the first complete purification of a rhamnosyltransferase. The enzyme from young pummelo leaves was purified >2,700-fold to a specific activity of over 600 pmol/min/mg of protein by sequential column chromatographies on Sephacryl S-200, reactive green 19-agarose, and Mono-Q. The enzyme was selectively eluted from the green dye column with only three other proteins by a pulse of the substrate hesperetin-7-O-glucoside followed by UDP. The rhamnosyltransferase is monomeric (∼52 kDa) by gel filtration and electrophoresis. The enzyme rhamnosylates only with UDP-rhamnose. Flavonoid-7-O-glucosides are usable acceptors but 5-O-glucosides or aglycones are not. It is inhibited by 10 μM UDP, its end product, but not by naringin or neohesperidin. Several flavonoid-aglycones at 100 μM inhibited the rhamnosyltransferase; UDP-sugars did not. The Km for UDP-rhamnose was similar with prunin (1.3 μM) and hesperetin-7-O-glucoside (1.1 μM) as substrate. The affinity for the natural acceptor prunin (Km = 2.4 μM) was much higher than for hesperetin-7-O-glucoside (Km = 41.5 μM). The isolation of the gene may enable its use in genetic engineering directed to modifying grapefruit bitterness.

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