An insect-specific P450 oxidative decarbonylase for cuticular hydrocarbon biosynthesis

Abstract

Insects use hydrocarbons as cuticular waterproofing agents and as contact pheromones. Although their biosynthesis from fatty acyl precursors is well established, the last step of hydrocarbon biosynthesis from long-chain fatty aldehydes has remained mysterious. We show here that insects use a P450 enzyme of the CYP4G family to oxidatively produce hydrocarbons from aldehydes. Oenocyte-directed RNAi knock-down of Drosophila CYP4G1 or NADPH-cytochrome P450 reductase results in flies deficient in cuticular hydrocarbons, highly susceptible to desiccation, and with reduced viability upon adult emergence. The heterologously expressed enzyme converts C 18 -trideuterated octadecanal to C 17 -trideuterated heptadecane, showing that the insect enzyme is an oxidative decarbonylase that catalyzes the cleavage of long-chain aldehydes to hydrocarbons with the release of carbon dioxide. This process is unlike cyanobacteria that use a nonheme diiron decarbonylase to make alkanes from aldehydes with the release of formate. The unique and highly conserved insect CYP4G enzymes are a key evolutionary innovation that allowed their colonization of land. Insects are the largest group of extant terrestrial organisms. As their crustacean ancestors moved from aquatic to terrestrial environments, insects were confronted with a new, dry frontier. Insects solved the ecophysiological problem of how to restrict water loss to prevent dessication by depositing long-chain hydrocarbons as essential waterproofing components on their epicuticle (1). These diverse chemicals now serve many additional functions, particularly in defense, reproduction, and communication (2). In flies, cuticular hydrocarbons (CHs) are a complex blend of long-chain (∼C21-C37+) alkanes and alkenes that serve as species-and sex-specific semiochemicals, and some components are sex pheromones. CHs also serve in nest mate recognition by social insects and as trail pheromones in ants; the complexity of their blend can be useful in taxonomic discrimination of mosquitoes. Much is known about the biosynthesis of CHs from fatty acids in insects, involving a complex network of fatty acid synthases, elongases, and desaturases, leading to very long-chain acyl-CoA thioesters. These are converted by acyl-CoA reductases to aldehydes that serve as substrates for the last oxidative decarbonylation step (2) (Fig. 1). The single carbon chain-shortening conversion of precursor aldehydes to hydrocarbons is catalyzed by a P450 enzyme, P450hyd, with release of CO 2 (3), but this enzyme has not yet been identified. The only known decarbonylase that has recently been described occurs in cyanobacteria that use a nonheme diiron enzyme (4-8) to catalyze the last step (Fig. 1). Insect CH are synthesized in large ectodermally derived cells (9) called oenocytes (10-12), and are then shuttled by hemolymph lipophorin (13,14) to the cuticle, where they are deposited on the outer epicuticular layer. Here we identify P450hyd as CYP4G1 in Drosophila melanogaster, and show that the enzyme is massively coexpresse...

Isolation and determination of absolute configurations of insect-produced methyl-branched hydrocarbons

“…Methyl-branched hydrocarbons isolated from various insect species and life stages, with measured specific rotations (±SD, n = 10 measurements) and absolute configurations. In all cases, the absolute configuration was (R) cytochrome-P450-mediated decarbonylation result in internally branched chiral MBCHs (2,56). Although the enoyl-ACP reductase domain has yet to be isolated from insects and there have been only a few reports of microsomal FAS isolation (54,57), FAS isolated from fungal species show highly conserved NADPH binding sites and genetic homology within the enoyl reductase domain (58), suggesting that this domain may also be conserved within the Insecta.…”

Dissecting ant recognition systems in the age of genomics

“…However, nearly all of these studies have been descriptive or correlational, and the biosynthetic pathways that underlie production and the mechanisms for generating molecular variation are not well understood. In fact, much of what we know about CHC synthesis in social insects relies on extrapolation from other, more well-studied, non-social insects, particularly species of Diptera [17][18][19][20].…”

“…Just as RNAi-mediated gene silencing has provided new insights into the molecular mechanisms that underlie chemical signalling in Drosophila (e.g. [20]), the application of these techniques to ants (and other social organisms) is likely to produce a wealth of new information about both the signal production and signal perception involved in colonymate recognition. These powerful new tools offer exciting opportunities to advance our understanding of Hamiltonian dynamics in the natural world, as we can now closely examine the interaction of the specific molecular players that are the phenotypic targets of kin-selected behaviours.…”

“…In Drosophila, oenocytedirected RNA interference (RNAi) was used to suppress gene expression of Cyp4g1 and (separately) NADPHcytochrome P450 reductase (CPR) [20]. In both cases, treated insects experienced high levels of mortality, and flies that did survive were characterized by radically altered CHC profiles.…”

“…Although the final biosynthetic step of CHC synthesis, the decarbonylation of long-chain fatty aldehydes to HCs, has long been believed to be catalysed by a P450 enzyme [18], the specific protein that performs this function has only recently been identified [20]. In Drosophila, oenocytedirected RNA interference (RNAi) was used to suppress gene expression of Cyp4g1 and (separately) NADPHcytochrome P450 reductase (CPR) [20].…”