title: Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases.
abstract: Membrane-bound desaturases are physiologically and industrially important enzymes that are involved in the production of diverse fatty acids such as polyunsaturated fatty acids and their derivatives. Here, we identified amino acid residues that determine the substrate specificity of rat Δ6 desaturase (D6d) acting on linoleoyl-CoA by comparing its amino acid sequence with that of Δ5 desaturase (D5d), which converts dihomo-γ-linolenoyl-CoA. The N-terminal cytochrome b5-like domain was excluded as a determinant by domain swapping analysis. Substitution of eight amino acid residues (Ser209, Asn211, Arg216, Ser235, Leu236, Trp244, Gln245, and Val344) of D6d with the corresponding residues of D5d by site-directed mutagenesis switched the substrate specificity from linoleoyl-CoA to dihomo-γ-linolenoyl-CoA. In addition, replacement of Leu323 of D6d with Phe323 on the basis of the amino acid sequence of zebra fish Δ5/6 bifunctional desaturase was found to render D6d bifunctional. Homology modeling of D6d using recent crystal structure data of human stearoyl-CoA (Δ9) desaturase revealed that Arg216, Trp244, Gln245, and Leu323 are located near the substrate-binding pocket. To our knowledge, this is the first report on the structural basis of the substrate specificity of a mammalian front-end fatty acid desaturase, which will aid in efficient production of value-added fatty acids.
authors.affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Japan.
authors.forename: Kenshi
authors.initials: K
authors.lastname: Watanabe
authors.affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ohno
authors.affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Taguchi
authors.affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Kawamoto
authors.affiliation: Department of Food Sciences and Biotechnology, Faculty of Life Sciences, Hiroshima Institute of Technology, Hiroshima, Japan.
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Ono
authors.affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Japan aki@hiroshima-u.ac.jp.
authors.forename: Tsunehiro
authors.initials: T
authors.lastname: Aki
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006801:Humans; D050602:Linoleoyl-CoA Desaturase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D005231:Fatty Acids, Unsaturated; D008565:Membrane Proteins; C017725:stearoyl-coenzyme A; C026562:linoleoyl-coenzyme A; D044943:Fatty Acid Desaturases; D050602:Linoleoyl-CoA Desaturase
keywords: heterologous expression; homology modeling; mass spectrometry; site-directed mutagenesis