Characterization of a recombinant 7,8-linoleate diol synthase from Glomerella cingulate

Appl Microbiol Biotechnol. 2016 Apr;100(7):3087-99. doi: 10.1007/s00253-015-7132-x. Epub 2015 Nov 18.

Abstract

A putative diol synthase from the fungus Glomerella cingulate was cloned and expressed in Escherichia coli. The putative diol synthase from G. cingulate was purified by His-Trap affinity chromatography with a specific activity of 0.87 U mg(-1), an eightfold purification, and a yield of 28%. One unit of activity was defined as the amount of enzyme required to produce 1 μmol of 7,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid (7,8-DiHODE) per min. The purified enzyme was estimated as a 127-kDa tetramer with a molecular mass of 510 kDa by gel filtration chromatography. The enzyme converted linoleic acid to a product, identified as 7S,8S-DiHODE by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The specific activity and catalytic efficiency (k cat/K m) of 7,8-diol synthase from G. cingulate for the conversion of fatty acid to dihydroxy fatty acid followed the order linoleic acid > α-linolenic acid > oleic acid > palmitoleic acid, indicating that the enzyme is a 7,8-linoleate diol synthase (7,8-LDS). The activity of the enzyme for the conversion of 7,8-DiHODE from linoleic acid was maximal at pH 6.5, 40 °C, and 2.5% (v/v) dimethyl sulfoxide (DMSO). Under these conditions, 7,8-LDS from G. cingulate converted 1.0 mM linoleic acid to 0.62 mM 7,8-DiHODE for 30 min, with a conversion yield of 62% (mol/mol), via 8-hydroperoxy-9,12(Z,Z)-octadecadienoic acid (8-HPODE) as an intermediate. The accumulation of 8-HPODE was due to a higher 8-dioxygenase activity in the N-terminal domain than hydroperoxide isomerase activity in the C-terminal domain.

Keywords: 7,8-Linoleate diol synthase; 7S,8S-Dihydroxy-9,12(Z,Z)-octadecadienoic acid; Enzyme characterization; Glomerella cingulate; Linoleic acid.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cloning, Molecular
  • Colletotrichum / chemistry*
  • Colletotrichum / enzymology
  • Dimethyl Sulfoxide / chemistry
  • Dimethyl Sulfoxide / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Fatty Acids, Monounsaturated / chemistry
  • Fatty Acids, Monounsaturated / metabolism
  • Fungal Proteins / chemistry*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Gene Expression
  • Hydrogen-Ion Concentration
  • Kinetics
  • Linoleic Acid / chemistry
  • Linoleic Acid / metabolism
  • Linoleic Acids / chemistry*
  • Linoleic Acids / metabolism
  • Molecular Weight
  • Oleic Acid / chemistry
  • Oleic Acid / metabolism
  • Oxygenases / chemistry*
  • Oxygenases / genetics
  • Oxygenases / metabolism
  • Protein Domains
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Substrate Specificity

Substances

  • Fatty Acids, Monounsaturated
  • Fungal Proteins
  • Linoleic Acids
  • Recombinant Proteins
  • 7,8-dihydroxylinoleic acid
  • palmitoleic acid
  • Oleic Acid
  • Linoleic Acid
  • Oxygenases
  • linoleate diol synthase
  • Dimethyl Sulfoxide