PUBLICATION
Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases
- Authors
- Watanabe, K., Ohno, M., Taguchi, M., Kawamoto, S., Ono, K., Aki, T.
- ID
- ZDB-PUB-151122-4
- Date
- 2016
- Source
- Journal of Lipid Research 57(1): 89-99 (Journal)
- Registered Authors
- Keywords
- Arachidonic acid, Enzymology, Enzymology/Enzyme mechanisms, Fatty acid/Biosynthesis, Fatty acid/Desaturases, heterologous expression, homology modeling, mass spectrometry, site-directed mutagenesis
- MeSH Terms
-
- Fatty Acid Desaturases/chemistry
- Fatty Acid Desaturases/genetics
- Fatty Acid Desaturases/metabolism*
- Amino Acid Sequence
- Acyl Coenzyme A/metabolism
- Fatty Acids, Unsaturated/metabolism
- Structure-Activity Relationship
- Saccharomyces cerevisiae/genetics
- Saccharomyces cerevisiae/metabolism
- Mutagenesis, Site-Directed/methods
- Linoleoyl-CoA Desaturase/chemistry
- Linoleoyl-CoA Desaturase/genetics
- Linoleoyl-CoA Desaturase/metabolism*
- Membrane Proteins/metabolism
- Molecular Sequence Data
- Substrate Specificity
- Cloning, Molecular
- Humans
- Models, Molecular
- Animals
- Escherichia coli/genetics
- Escherichia coli/metabolism
- Rats
- PubMed
- 26590171 Full text @ J. Lipid Res.
Citation
Watanabe, K., Ohno, M., Taguchi, M., Kawamoto, S., Ono, K., Aki, T. (2016) Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases. Journal of Lipid Research. 57(1):89-99.
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 ∆6 desaturase (D6d) acting on linoleoyl-CoA by comparing its amino acid sequence with that of ∆5 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 zebrafish ∆5/6 bifunctional desaturase was found to render D6d bifunctional. Homology modeling of D6d using recent crystal structure data of human stearoyl-CoA (∆9) 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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping