title: Characterization of a recombinant 7,8-linoleate diol synthase from Glomerella cingulate.
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.
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, Republic of Korea.
authors.forename: Min-Ju
authors.initials: MJ
authors.lastname: Seo
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, Republic of Korea.
authors.forename: Kyung-Chul
authors.initials: KC
authors.lastname: Shin
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, Republic of Korea.
authors.forename: Jung-Ung
authors.initials: JU
authors.lastname: An
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, Republic of Korea.
authors.forename: Woo-Ri
authors.initials: WR
authors.lastname: Kang
authors.affiliation: National Center for Inter-University Research Facilities (NCIRF), Seoul National University, Seoul, 08826, Republic of Korea.
authors.forename: Yoon-Joo
authors.initials: YJ
authors.lastname: Ko
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, Republic of Korea. deokkun@konkuk.ac.kr.
authors.forename: Deok-Kun
authors.initials: DK
authors.lastname: Oh
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D020231:Colletotrichum / Q000737:chemistry* / Q000201:enzymology; D004121:Dimethyl Sulfoxide / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019787:Linoleic Acid / Q000737:chemistry / Q000378:metabolism; D008041:Linoleic Acids / Q000737:chemistry* / Q000378:metabolism; D008970:Molecular Weight; D019301:Oleic Acid / Q000737:chemistry / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D005229:Fatty Acids, Monounsaturated; D005656:Fungal Proteins; D008041:Linoleic Acids; D011994:Recombinant Proteins; C075808:7,8-dihydroxylinoleic acid; C008757:palmitoleic acid; D019301:Oleic Acid; D019787:Linoleic Acid; D010105:Oxygenases; C122014:linoleate diol synthase; D004121:Dimethyl Sulfoxide
keywords: 7,8-Linoleate diol synthase; 7S,8S-Dihydroxy-9,12(Z,Z)-octadecadienoic acid; Enzyme characterization; Glomerella cingulate; Linoleic acid