Purification and characterization of p-coumaroyl-D-glucose hydroxylase of sweet potato (Ipomoea batatas) roots

Arch Biochem Biophys. 1991 Jan;284(1):151-7. doi: 10.1016/0003-9861(91)90277-p.

Abstract

p-Coumaroyl-D-glucose hydroxylase in sweet potato (Ipomoea batatas Lam.) has been purified to apparent electrophoretic homogeneity using a combination of anion-and cation-exchange, hydrophobic and gel filtration chromatography. The purified enzyme was a monomer with a molecular weight of 33,000 and pI of 8.3. The purified enzyme showed not only hydroxylase activity but also polyphenol oxidase activity. L-Ascorbic acid was the best electron donor for the hydroxylation reaction, which had an optimum pH of 7.0. The enzyme hydroxylated p-coumaroyl-D-glucose, p-coumaric acid, and p-cresol but did not act on o-coumaric acid, m-coumaric acid, 4-hydroxy-3-methoxycinnamic acid, p-hydroxybenzoic acid or L-tyrosine. While the enzyme utilized p-coumaroyl-D-glucose and p-coumaric acid equally at pH 7.0, it hydroxylated only p-coumaroyl-D-glucose at pH 5.5. The enzyme oxidized diphenols such as D,L-(3,4-dihydroxyphenyl) alanine and caffeic acid, but exhibited no clear pH optimum in this reaction characteristic of polyphenol oxidase. Both the hydroxylase and the polyphenol oxidase activities were strongly inhibited by beta-mercaptoethanol, diethyldithiocarbamate, KCN, and p-coumaric acid (in concentrations higher than 5 mM). Ammonium sulfate and sodium chloride activated the hydroxylase activity but not the polyphenol oxidase activity of the enzyme. The enzyme activity and L-ascorbic acid contents changed in a manner suggesting their involvements in chlorogenic acid biosynthesis during incubation of sliced sweet potato root tissues.

MeSH terms

  • Ammonium Sulfate / pharmacology
  • Ascorbic Acid / metabolism
  • Chlorogenic Acid / metabolism
  • Coumaric Acids / pharmacology
  • Flavonoids*
  • Hydrogen-Ion Concentration
  • Hydrolases / antagonists & inhibitors
  • Hydrolases / chemistry
  • Hydrolases / isolation & purification*
  • Hydrolases / metabolism
  • Isoelectric Point
  • Mixed Function Oxygenases*
  • Molecular Weight
  • Phenols / metabolism
  • Plants / enzymology*
  • Polymers / metabolism
  • Polyphenols
  • Propionates
  • Sodium Chloride / pharmacology
  • Substrate Specificity

Substances

  • Coumaric Acids
  • Flavonoids
  • Phenols
  • Polymers
  • Polyphenols
  • Propionates
  • Chlorogenic Acid
  • Sodium Chloride
  • Mixed Function Oxygenases
  • 4-coumaroyl-D-glucose hydroxylase
  • Hydrolases
  • trans-3-(4'-hydroxyphenyl)-2-propenoic acid
  • Ascorbic Acid
  • Ammonium Sulfate