Unexpected functional diversity in the fatty acid desaturases of the flour beetle Tribolium castaneum and identification of key residues determining activity

摘要

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 Delta 9 desaturases, second clade was Tribollum-specific having diverse activities including Delta 5, Delta 9 and Delta 12 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 Delta 12 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: Delta 9 desaturation of C12:0 C16:0 substrates; two unprecedented Delta 5 enzymes acting on C18:0 and C16:0; Delta 9 activity exclusively on C16:0 and a further stearate Delta 9 desaturase. qPCR analysis ruled out a role in sex pheromone synthesis for the Delta 5 and Delta 9/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. Crown Copyright (C) 2014 Published by Elsevier Ltd. All rights reserved.