title: Unexpected functional diversity in the fatty acid desaturases of the flour beetle Tribolium castaneum and identification of key residues determining activity.
abstract: Desaturases catalyse modifications to fatty acids which are essential to homeostasis and for pheromone and defensive chemical production. All desaturases of the flour beetle Tribolium castaneum were investigated via query of the sequenced genome which yielded 15 putative acyl-Coenzyme A genes. Eleven desaturase mRNA were obtained in full length and functionally expressed in yeast. Phylogenetic analysis separated the desaturases into 4 distinct clades; one clade contained conserved beetle Δ9 desaturases, second clade was Tribolium-specific having diverse activities including Δ5, Δ9 and Δ12 desaturation and the other 2 clades had mixed insect representatives. Three members of this clade contained unusual inserted sequences of ∼20 residues in the C-terminal region and were related to desaturases that all contained similar inserts. Deletion of the entirety of the insert in the flour beetle Δ12 desaturase abolished its activity but this was partially restored by the reintroduction of two histidine residues, suggesting the histidine(s) are required for activity but the full length insert is not. Five new desaturase activities were discovered: Δ9 desaturation of C12:0-C16:0 substrates; two unprecedented Δ5 enzymes acting on C18:0 and C16:0; Δ9 activity exclusively on C16:0 and a further stearate Δ9 desaturase. qPCR analysis ruled out a role in sex pheromone synthesis for the Δ5 and Δ9/C16:0 desaturases. The flour beetle genome has underpinned an examination of all transcribed desaturases in the organism and revealed a diversity of novel and unusual activities, an improved understanding of the evolutionary relationships among insect desaturases and sequence determinants of activity.
authors.affiliation: CSIRO Ecosystem Sciences, GPO Box 1700, Canberra, ACT, 2601 Australia. Electronic address: victoria.haritos@csiro.au.
authors.forename: Victoria S
authors.initials: VS
authors.lastname: Haritos
authors.affiliation: CSIRO Ecosystem Sciences, GPO Box 1700, Canberra, ACT, 2601 Australia.
authors.forename: Irene
authors.initials: I
authors.lastname: Horne
authors.affiliation: CSIRO Ecosystem Sciences, GPO Box 1700, Canberra, ACT, 2601 Australia.
authors.forename: Katherine
authors.initials: K
authors.lastname: Damcevski
authors.affiliation: CSIRO Ecosystem Sciences, GPO Box 1700, Canberra, ACT, 2601 Australia.
authors.forename: Karen
authors.initials: K
authors.lastname: Glover
authors.affiliation: CSIRO Ecosystem Sciences, GPO Box 1700, Canberra, ACT, 2601 Australia.
authors.forename: Nerida
authors.initials: N
authors.lastname: Gibb
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence / Q000235:genetics; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry; D008969:Molecular Sequence Data; D010675:Pheromones / Q000096:biosynthesis; D010802:Phylogeny; D012441:Saccharomyces cerevisiae; D012724:Sex Attractants / Q000096:biosynthesis; D013230:Stearoyl-CoA Desaturase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014231:Tribolium / Q000201:enzymology* / Q000235:genetics*
chemical_list: D005227:Fatty Acids; D010675:Pheromones; D012724:Sex Attractants; D044943:Fatty Acid Desaturases; D013230:Stearoyl-CoA Desaturase
keywords: Acyl-coenzyme A desaturases; Gene expression; Membrane enzymes; Phylogenetics; Unsaturated fatty acid