Isolation and characterization of a lipoxygenase from Pseudomonas 42A2 responsible for the biotransformation of oleic acid into ( S )-( E )-10-hydroxy-8-octadecenoic acid

Antonie Van Leeuwenhoek. 2004 Feb;85(2):129-39. doi: 10.1023/B:ANTO.0000020152.15440.65.

Abstract

The isolation of a new lipoxygenase-like (LOX-like) enzyme from Pseudomonas 42A2 and its characterization is described. The enzyme, located in the periplasm of the cell, which contained 0.55 mol of Fe2+ per mol of protein, is monomeric and has a molecular mass of 45 kDa. In the presence of oxygen, the enzyme converts oleic acid into (E)-10-hydroperoxy-8-octadecenoic acid (HPOD), which decomposes to the corresponding (E)-10-hydroxy-8-octadecenoic acid (HOD). The absolute configuration of this acid was determined as S on the basis of exciton-coupled CD data, and specific rotation and NMR analysis of the corresponding p -bromobenzoate derivative. The reaction in vivo leads to the dihydroxy derivative (E)-7,10-dihydroxy-8-octadecenoic acid (DHOD), so that the three hydroxy-fatty acids can be isolated from the culture medium. The activity of the enzyme was optimal between 25 and 30 degrees C and 44% of its activity still remained at 55 degrees C. Its optimal pH is 8.5-9; and the presence of magnesium ions increased LOX activity by 1.5. The activity of the LOX is highest in unsaturated fatty acids containing double bonds in position 9 (oleic, linoleic and linolenic acids), linoleic acid being preferred (100% activity) over linolenic (60.4%) and oleic acids (46%). However, kinetic studies showed that the affinity of the enzyme is similar for the three substrates.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotransformation
  • Cell Fractionation
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Stability
  • Hydrogen-Ion Concentration
  • Kinetics
  • Lipoxygenase / chemistry
  • Lipoxygenase / isolation & purification*
  • Lipoxygenase / metabolism*
  • Oleic Acid / metabolism*
  • Oleic Acids / metabolism
  • Pseudomonas / enzymology*
  • Thermodynamics

Substances

  • 10-hydroxy-8-octadecenoic acid
  • Oleic Acids
  • Oleic Acid
  • Lipoxygenase