Functional characterization of a desaturase from the tobacco hornworm moth (Manduca sexta) with bifunctional Z11- and 10,12-desaturase activity

Matoušková, Petra; Pichová, Iva; Svatoš, Aleš

doi:10.1016/j.ibmb.2007.03.004

Cited by 36 publications

(29 citation statements)

References 28 publications

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“…To select candidate FAD genes involved in pheromone biosynthesis, we performed RNA sequencing of M. sexta female PGs, the site of pheromone biosynthesis (19), as well as nonpheromone-producing tissues (female fat body, female labial palps, and larval midgut). We identified 14 desaturase transcripts, of which 4 were abundant and enriched in the PG according to normalized expression values (RPKM, reads per kilobase of transcript per million mapped reads): MsexD2, a previously characterized Z11-desaturase/conjugase involved in sequential biosynthesis of 1UFA and 2UFA pheromone precursors (18), and three FAD gene products, MsexD3, MsexD5 and MsexD6 (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…Using next-generation sequencing of M. sexta female PG and reference tissues, we identified a set of highly abundant and PGenriched FAD transcripts, including MsexD2, a previously described FAD involved in biosynthesis of mono-and diunsaturated SPC precursors (18), and previously unidentified MsexD3, MsexD5, and MsexD6 FADs. The 3UFA precursor of the essential M. sexta SPC E10,E12,Z14-16:3-aldehyde (27), along with E10,E12,E14-16:3, was produced by MsexD3 and MsexD5 in our yeast expression system.…”

Section: Discussionmentioning

confidence: 99%

“…In our previous attempts to decipher the desaturation pathway leading to triunsaturated SPC FA precursors (3UFAs), we identified the MsexD2 desaturase, which exhibits Z11-desaturase and conjugase (1,4-dehydrogenase) activity and participates in stepwise production of monounsaturated (1UFA) and diunsaturated (2UFA) SPC precursors. The terminal desaturation step resulting in the third conjugated double bond remained, however, elusive (18,19).…”

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confidence: 99%

“…Heterologous FAD expression in pYEXTHS-BN transformed yeasts was monitored by Western blotting using anti-His6tag antibodies (37). Total cellular lipids were transesterified to FAMEs (18). When indicated, yeast strains were cultivated with FAMEs supplemented to a final concentration of 0.25 mM in cultivation medium.…”

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

Buček

Matoušková

Vogel

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Buček

Matoušková

Vogel

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…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].…”

Section: Mfad Propertiesmentioning

confidence: 99%

Biotechnological potential of insect fatty acid-modifying enzymes

Tupec

Buček

Valterová

2017

Zeitschrift Für Naturforschung C

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There are more than one million described insect species. This species richness is reflected in the diversity of insect metabolic processes. In particular, biosynthesis of secondary metabolites, such as defensive compounds and chemical signals, encompasses an extraordinarily wide range of chemicals that are generally unparalleled among natural products from other organisms. Insect genomes, transcriptomes and proteomes thus offer a valuable resource for discovery of novel enzymes with potential for biotechnological applications. Here, we focus on fatty acid (FA) metabolism-related enzymes, notably the fatty acyl desaturases and fatty acyl reductases involved in the biosynthesis of FA-derived pheromones. Research on insect pheromone-biosynthetic enzymes, which exhibit diverse enzymatic properties, has the potential to broaden the understanding of enzyme specificity determinants and contribute to engineering of enzymes with desired properties for biotechnological production of FA derivatives. Additionally, the application of such pheromone-biosynthetic enzymes represents an environmentally friendly and economic alternative to the chemical synthesis of pheromones that are used in insect pest management strategies.

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Regulation of Isoprenoid Pheromone Biosynthesis in Bumblebee Males

et al. 2015

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Males of the closely related species Bombus terrestris and Bombus lucorum attract conspecific females by completely different marking pheromones. MP of B. terrestris and B. lucorum pheromones contain mainly isoprenoid (ISP) compounds and fatty acid derivatives, respectively. Here, we studied the regulation of ISP biosynthesis in both bumblebees. RNA-seq and qRT-PCR analyses indicated that acetoacetyl-CoA thiolase (AACT), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), and farnesyl diphosphate synthase (FPPS) transcripts are abundant in the B. terrestris labial gland. Maximal abundance of these transcripts correlated well with AACT enzymatic activity detected in the LG extracts. In contrast, transcript abundances of AACT, HMGR, and FPPS in B. lucorum were low, and AACT activity was not detected in LGs. These results suggest that transcriptional regulation plays a key role in the control of ISP biosynthetic gene expression and ISP pheromone biosynthesis in bumblebee males.

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Functional characterization of a desaturase from the tobacco hornworm moth (Manduca sexta) with bifunctional Z11- and 10,12-desaturase activity

Cited by 36 publications

References 28 publications

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

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

Biotechnological potential of insect fatty acid-modifying enzymes

Regulation of Isoprenoid Pheromone Biosynthesis in Bumblebee Males

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