Cloning and functional expression of a cDNA encoding a pheromone gland-specific acyl-CoA Δ ¹¹ -desaturase of the cabbage looper moth, Trichoplusia ni

Abstract

Desaturation of coenzyme-A esters of saturated fatty acids is a common feature of sex pheromone biosynthetic pathways in the Lepidoptera. The enzymes that catalyze this step share several biochemical properties with the ubiquitous acyl-CoA Δ 9 -desaturases of animals and fungi, suggesting a common ancestral origin. Unlike metabolic acyl-CoA Δ 9 -desaturases, pheromone desaturases have evolved unusual regio- and stereoselective activities that contribute to the remarkable diversity of chemical structures used as pheromones in this large taxonomic group. In this report, we describe the isolation of a cDNA encoding a pheromone gland desaturase from the cabbage looper moth, Trichoplusia ni , a species in which all unsaturated pheromone products are produced via a Δ 11 Z-desaturation mechanism. The largest ORF of the ≈1,250-bp cDNA encodes a 349-aa apoprotein (PDesat-Tn Δ 11 Z) with a predicted molecular mass of 40,240 Da. Its hydrophobicity profile is similar overall to those of rat and yeast Δ 9 -desaturases, suggesting conserved transmembrane topology. A 182-aa core domain delimited by conserved histidine-rich motifs implicated in iron-binding and catalysis has 72 and 58% similarity (including conservative substitutions) to acyl-CoA Δ 9 Z-desaturases of rat and yeast, respectively. Northern blot analysis revealed an ≈1,250-nt PDesat-Tn Δ 11 Z mRNA that is consistent with the spatial and temporal distribution of Δ 11 -desaturase enzyme activity. Genetic transformation of a desaturase-deficient strain of the yeast Saccharomyces cerevisiae with an expression plasmid encoding PDesat-Tn Δ 11 Z resulted in complementation of the strain’s fatty acid auxotrophy and the production of Δ 11 Z-unsaturated fatty acids.

A multifunctional desaturase involved in the biosynthesis of the processionary moth sex pheromone

“…5H), which produced the C11-monounsaturated hexa-and tetradecenoates, but did not make the corresponding C18 compound when expressed in yeast by using the pYEXTHS-BN vector (19). In contrast, (Z)-11-octadecenoic acid is synthesized by yeast transformed with either pYEXTHS-BN or the YEpOLEXbased plasmids containing the T. ni ⌬ 11 desaturase gene (5,19). These differences suggest subtle dissimilarities in the tridimensional structure of the enzyme active sites, which result in different capabilities for accommodating long acyl substrates.…”

“…The desaturases expressed in female sex-pheromone glands of different moth species have evolved to play a key role in the biosynthesis of sex pheromones, exhibiting an amazing diversity of substrate and regio-and stereospecifities. Unlike the metabolic ⌬ 9 acyl-CoA desaturases, pheromone gland desaturases catalyze the formation of uncommon unsaturated fatty acyl-CoA esters with variable chain lengths, different locations of unsaturations, and either the ordinary Z or the unusual E double bond geometry (3)(4)(5)(6)(7)(8)(9)(10).…”

Molecular genetics and evolution of pheromone biosynthesis in Lepidoptera

“…The structures of ⌬9 and ⌬11 desaturase-encoding cDNAs from T. ni and the corn earworm moth, Helicoverpa zea, were first characterized and reported (8)(9)(10). Research on other selected species resulted in the characterization of structures of other pheromone desaturases with different regio-and stereospecificities, including Z͞E11, E11, Z10, Z9, and Z͞E14 desaturases (8)(9)(10)(11)(12)(13)(14)(15).…”

“…Research on other selected species resulted in the characterization of structures of other pheromone desaturases with different regio-and stereospecificities, including Z͞E11, E11, Z10, Z9, and Z͞E14 desaturases (8)(9)(10)(11)(12)(13)(14)(15). Cloning studies using mRNA isolated from fat bodies and pheromone glands revealed that there were at least two classes of ⌬9 desaturases in the Lepidoptera.…”

“…This proved to be difficult with an in vitro recon-stitutive biochemical assay because integral membrane desaturases were shown to use a complex consisting of the desaturase protein, NADH-cytochrome b 5 reductase (a flavoprotein), and cytochrome b 5 (a hemoprotein). However, a discovery that the yeast OLE1 desaturase can be functionally replaced in vivo by the rat desaturase (7) led the way to the development of yeast expression systems that could be used for the functional assay of pheromone desaturases (8).…”