UDP-rhamnose:flavanone-7-O-glucoside-2''-O-rhamnosyltransferase. Purification and Characterization of an Enzyme Catalyzing the Production of Bitter Compounds in Citrus

J Biol Chem. 1991 Nov 5;266(31):20953-9.

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 rhamnosyl-transferase. The enzyme from young pummelo leaves was purified greater than 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 (approximately 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 microM UDP, its end product, but not by naringin or neohesperidin. Several flavonoid-aglycones at 100 microM inhibited the rhamnosyltransferase; UDP-sugars did not. The Km for UDP-rhamnose was similar with prunin (1.3 microM) and hesperetin-7-O-glucoside (1.1 microM) as substrate. The affinity for the natural acceptor prunin (Km = 2.4 microM) was much higher than for hesperetin-7-O-glucoside (Km = 41.5 microM). The isolation of the gene may enable its use in genetic engineering directed to modifying grapefruit bitterness.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography
  • Flavonoids / metabolism*
  • Fruit / enzymology*
  • Hexosyltransferases / isolation & purification*
  • Molecular Weight
  • Plant Proteins / chemistry
  • Plant Proteins / isolation & purification*

Substances

  • Flavonoids
  • Plant Proteins
  • Hexosyltransferases
  • UDP-rhamnose-flavanone-7-O-glucoside-2''-O-rhamnosyltransferase