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.

Fig. 1.

Hydrocarbon biosynthesis from very long-chain fatty acyl thioesters in cyanobacteria and in insects. The decarbonylase enzyme from plants has not been formally identified to date. ACP, acyl carrier protein.

Fig. 2.

Colocalization of CYP4G1 and CPR in oenocytes. Whole-mount immunocytochemistry of NADPH-cytochrome P450 reductase (Upper Left, FITC) and CYP4G1 (Upper Right, Alexa 633) in Drosophila abdomens. Confocal microscopy shows the bands of large oenocytes where both enzymes are colocalized (Lower Right, yellow). Lower Left is the bright field image showing bristles for scale.

Fig. 3.

Effects of RNAi suppression of CYP4G1 and CPR on cuticular hydrocarbons. Principal component analysis (PCA) of the peaks identified from the GC-MS profile of cuticle hexane washes of 1-d posteclosion flies. The correlation matrix of the values in nanograms per fly for 38 peaks and 107 profiles corresponding to five genotypes was analyzed using the PRINCOMP procedure in SAS (v9). The first two axes of the PCA account for 62.8% of the total variance in the data. Each ellipse represents the 2D 95% confidence interval of the mean of data for five fly genotypes. The ellipses on the right are the three parental genotypes (OE-GAL4, oenocyte GAL4 driver; UAS-4G1, UAS-dsRNA for CYP4G1; UAS-CPR, UAS-dsRNA for CPR) (n = 12, 10, and 10, respectively). On the left are the two GAL4-UAS offspring genotypes, 4G1-RNAi and CPR-RNAi (n = 39 and 36, respectively), that are similar to each other and nonoverlapping with their parents. The histograms on the right are the mean value of each peak for the five genotypes on the same scale. The peaks shown are 12 esters and fatty acids to the left and 26 hydrocarbons to the right, ranked by molecular weight (details in Table S1).

Fig. 4.

Desiccation resistance of adult D. melanogaster. The time course of adult male (▲) and female (●) fly survival in dry conditions is shown for control insects (full lines) and for flies with RNAi-suppressed CYP4G1 expression (stippled lines). n = 20 for each condition.

Fig. 5.

Metabolism of aldehyde to alkane by recombinant CYP4G2/CPR fusion enzyme. Mass spectrum of trideuterated reaction product from [18,18,18-²H₃]octadecanal (A) and of reference heptadecane (B). (Inset) Trapping assays show the carbonyl-carbon of [1-¹⁴C]-C18 aldehyde is released as CO₂. Values are means ± SEM, n = 3. BG, background.