Evolution of moth sex pheromone composition by a single amino acid substitution in a fatty acid desaturase

Abstract

For sexual communication, moths primarily use blends of fatty acid derivatives containing one or more double bonds in various positions and configurations, called sex pheromones (SPs). To study the molecular basis of novel SP component (SPC) acquisition, we used the tobacco hornworm (Manduca sexta), which uses a blend of mono-, di-, and uncommon triunsaturated fatty acid (3UFA) derivatives as SP. We identified pheromone-biosynthetic fatty acid desaturases (FADs) MsexD3, MsexD5, and MsexD6 abundantly expressed in the M. sexta female pheromone gland. Their functional characterization and in vivo application of FAD substrates indicated that MsexD3 and MsexD5 biosynthesize 3UFAs via E/Z14 desaturation from diunsaturated fatty acids produced by previously characterized Z11-desaturase/conjugase MsexD2. Site-directed mutagenesis of sequentially highly similar MsexD3 and MsexD2 demonstrated that swapping of a single amino acid in the fatty acyl substrate binding tunnel introduces E/Z14-desaturase specificity to mutated MsexD2. Reconstruction of FAD gene phylogeny indicates that MsexD3 was recruited for biosynthesis of 3UFA SPCs in M. sexta lineage via gene duplication and neofunctionalization, whereas MsexD5 representing an alternative 3UFA-producing FAD has been acquired via activation of a presumably inactive ancestral MsexD5. Our results demonstrate that a change as small as a single amino acid substitution in a FAD enzyme might result in the acquisition of new SP compounds.fatty acid desaturase | Manduca sexta | sex pheromone biosynthesis | pheromone evolution | substrate specificity S ex pheromones (SPs) are a diverse group of chemical compounds that are central to mate-finding behavior in insects (1). Variation in SP composition between closely related species and among populations is well documented. Despite this variation, SPs are presumed to be under strong stabilizing selection, and thus the genetic mechanisms driving SP diversification represented an enigma (2). Research on SPs in moths (Insecta: Lepidoptera) helped establish the hypothesis of asymmetric tracking as a major driving force in SP diversification. In this scenario, abrupt changes in female SP composition via a shift in component ratio or the inclusion or loss of a component result in a distinct SP that attracts males with more broadly or differentially tuned SP preference (3). Assortative mating, the preferential mating of females producing a novel SP with males attracted to this SP, restricts gene flow between subpopulations with differing SP compositions. This can ultimately lead to speciation and fixation of novel communication channels (4). Work in insect models such as wasps (5), fruit flies (6), and especially moths (7-9) is helping uncover the genetic basis of SP diversification.In the majority of moth species, females use species-specific mixtures of SP components (SPCs) consisting of volatile fatty acid (FA) derivatives to attract conspecific males at long range. These SPCs are predominantly long-chain aliphatic (C12-C18) acetates, alcoh...

Biotechnological potential of insect fatty acid-modifying enzymes

“…The preferred fatty acyl chain length may be C14 [27,[66][67][68][70][71][72][73], C16 [74], or C18 [63,75,76], but some mFADs can desaturate a broad range of fatty acyl chain lengths, such as C14-C20 [67,77]. The positions of the introduced double bond include ∆9, ∆4 [33], ∆5 [72], ∆6 [64,78], ∆8 [65], ∆10 [69,78], ∆11 [10, 63, 65-67, 74-76, 79, 80], ∆13 [81], and ∆14 [70,82]. Δ12 mFADs have also been identified, but they are involved in primary metabolism during the production of methylene-interrupted PUFAs [31] rather than pheromone biosynthesis.…”

“…Δ12 mFADs have also been identified, but they are involved in primary metabolism during the production of methylene-interrupted PUFAs [31] rather than pheromone biosynthesis. The moth mFADs can also introduce a double bond at the terminal position between the penultimate and ultimate carbon atoms [78,83] and can produce FAs with a system of isolated double bonds [64] or a system of conjugated double bonds [10,65,66,76,79,80,82]. Among the more bizarre desaturation reactions, an mFAD identified in the processionary moth, Thaumetopoea pityocampa, can introduce triple bonds into the FA chains [81], resembling in activity an mFAD described in the plant of Crepis genus [84].…”

“…The process of functional divergence of pheromone-biosynthetic enzymes, which can be observed in insect subpopulations or closely related species, generates biocatalysts that are highly similar in protein sequence yet distinct in their enzymatic properties. These enzymes are convenient model systems to study the mechanisms of enzyme specificity determinants [82,119,150]. Importantly, the amassed knowledge about the function of the FA-modifying enzymes could be used to design novel enzymes with desired enzymatic properties.…”

“…These experiments led to the identification of sequence determinants of both acyl-CoA and acyl-lipid mFAD specificities in diverse organisms, such as transmembrane helices or conserved histidine-rich motifs [55,59,[152][153][154][155][156][157][158][159][160][161][162]. Buček et al [82] identified a critical amino acid residue in the transmembrane domain of an mFAD from Manduca sexta (MsexD3) that determines the specificity and ability of this desaturase to catalyze biosynthesis of FAs containing three conjugated double bonds via E/Z14 desaturation from diunsaturated FAs.…”

“…Indeed, the homology model of MsexD3 highlighted the prominent position of the critical residue Ile224 in the kink of the fatty acyl substrate binding tunnel, which presumably plays a critical role in positioning the substrate fatty acyl chain with respect to the di-iron active center [82]. Ding et al found that reciprocal exchange of a single amino acid residue in moth ∆11 mFADs can switch between their E and Z desaturase specificities.…”