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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.