title: Identification of amino acids conferring chain length substrate specificities on fatty alcohol-forming reductases FAR5 and FAR8 from Arabidopsis thaliana.
abstract: Fatty alcohols play a variety of biological roles in all kingdoms of life. Fatty acyl reductase (FAR) enzymes catalyze the reduction of fatty acyl-coenzyme A (CoA) or fatty acyl-acyl carrier protein substrates to primary fatty alcohols. FAR enzymes have distinct substrate specificities with regard to chain length and degree of saturation. FAR5 (At3g44550) and FAR8 (At3g44560) from Arabidopsis thaliana are 85% identical at the amino acid level and are of equal length, but they possess distinct specificities for 18:0 or 16:0 acyl chain length, respectively. We used Saccharomyces cerevisiae as a heterologous expression system to assess FAR substrate specificity determinants. We identified individual amino acids that affect protein levels or 16:0-CoA versus 18:0-CoA specificity by expressing in yeast FAR5 and FAR8 domain-swap chimeras and site-specific mutants. We found that a threonine at position 347 and a serine at position 363 were important for high FAR5 and FAR8 protein accumulation in yeast and thus are likely important for protein folding and stability. Amino acids at positions 355 and 377 were important for dictating 16:0-CoA versus 18:0-CoA chain length specificity. Simultaneously converting alanine 355 and valine 377 of FAR5 to the corresponding FAR8 residues, leucine and methionine, respectively, almost fully converted FAR5 specificity from 18:0-CoA to 16:0-CoA. The reciprocal amino acid conversions, L355A and M377V, made in the active FAR8-S363P mutant background converted its specificity from 16:0-CoA to 18:0-CoA. This study is an important advancement in the engineering of highly active FAR proteins with desired specificities for the production of fatty alcohols with industrial value.
authors.affiliation: From the Institute of Biochemistry, Department of Biology, Carleton University, Ottawa, Ontario K1S 5B6, Canada and.
authors.forename: Micaëla G
authors.initials: MG
authors.lastname: Chacón
authors.affiliation: From the Institute of Biochemistry, Department of Biology, Carleton University, Ottawa, Ontario K1S 5B6, Canada and.
authors.forename: Ashley E
authors.initials: AE
authors.lastname: Fournier
authors.affiliation: From the Institute of Biochemistry, Department of Biology, Carleton University, Ottawa, Ontario K1S 5B6, Canada and.
authors.forename: Frances
authors.initials: F
authors.lastname: Tran
authors.affiliation: the Laboratoire de Biogenèse Membranaire, Université Bordeaux Ségalen, CNRS-UMR 5200, Bâtiment A3-INRA Bordeaux Aquitaine BP81, 71 Avenue Edouard Bourlaux, 33883 Villenave D'Ornon Cedex, France.
authors.forename: Franziska
authors.initials: F
authors.lastname: Dittrich-Domergue
authors.affiliation: From the Institute of Biochemistry, Department of Biology, Carleton University, Ottawa, Ontario K1S 5B6, Canada and.
authors.forename: Ian P
authors.initials: IP
authors.lastname: Pulsifer
authors.affiliation: the Laboratoire de Biogenèse Membranaire, Université Bordeaux Ségalen, CNRS-UMR 5200, Bâtiment A3-INRA Bordeaux Aquitaine BP81, 71 Avenue Edouard Bourlaux, 33883 Villenave D'Ornon Cedex, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Domergue
authors.affiliation: From the Institute of Biochemistry, Department of Biology, Carleton University, Ottawa, Ontario K1S 5B6, Canada and. Electronic address: owen_rowland@carleton.ca.
authors.forename: Owen
authors.initials: O
authors.lastname: Rowland
mesh_terms: D000214:Acyl Coenzyme A / Q000235:genetics / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability / Q000502:physiology; D015870:Gene Expression; D020125:Mutation, Missense; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D000214:Acyl Coenzyme A; D029681:Arabidopsis Proteins; D011994:Recombinant Proteins; D000445:Aldehyde Oxidoreductases; C021351:hexadecanal dehydrogenase (acylating)
keywords: Enzyme Structure; Fatty Acyl Reductase; Fatty Alcohol; Lipid Metabolism; Plant Biochemistry; Protein Engineering; Site-directed Mutagenesis