Aldehyde Decarbonylases: Enigmatic Enzymes of Hydrocarbon Biosynthesis

Long-chain alkane waxes are synthesized by a wide variety of organisms, including plants, animals and microorganisms.1-4 Although chemically alkanes are amongst the most boring of organic molecules, devoid of functionality and thus unreactive, they play essential roles in these organisms’ survival. Plants secrete what are termed “very long chain” alkane waxes (i.e. chain length ~ 30 carbons) onto their leaves and stems.3 These, together with wax esters formed from aliphatic alcohols and carboxylic acids of similar chain length, serve as a waterproof barrier to prevent desiccation. A wide variety of hydrocarbons, including branched and unsaturated molecules, are biosynthesized by insects.2 These are secreted onto the cuticle and play an important role as contact pheromones that mediate all manner of insect:insect recognition and social behaviors; they are also important in preventing desiccation, especially of larval forms.5 Waterfowl secrete alkane-rich oils from their uropygial (preening) glands; these are essential for waterproofing the birds’ feathers, which would otherwise become waterlogged and thus useless for insulation and flight.1 Many algae synthesize large amounts of long-chain alkanes that can comprise up to 30 % of the algae's dry weight.6 These accumulate in the area contained by the inner and outer cell walls known as the trilamellar structure and can be utilized to provide energy when photosynthesis is not possible.7 In recent decades many human beings have become equally dependent on alkanes, in that this class of molecules is the major component of transportation fuels. These fuels are, of course, currently derived from fossil reserves and their combustion is a major source of greenhouse gases. The challenge of producing the next generation of biofuels – those that can effectively function as “drop-in” replacements for gasoline, diesel, and jet fuel – has led to renewed interest, especially in the area of mechanistic enzymology, in how alkanes are biosynthesized.8 The synthesis of completely unfunctionalized molecules presents a challenge for a cell.9 Whereas as a chemist might accomplish this by reducing the corresponding alkene to an alkane using hydrogen and an activated transition metal catalyst, this approach is clearly not biochemically feasible. Historically, an important observation was that naturally-synthesized alkanes invariably comprise an odd number of carbons, suggesting that they are derived from fatty acid biosynthesis through loss of the carboxyl carbon.10 It was subsequently established that long-chain alkanes are synthesized from fatty acids through the intermediacy of the corresponding fatty aldehydes. These are the substrates for a group of enzymes, the aldehyde decarbonylases, which catalyze the removal of the aldehyde carbonyl group to form the alkane. It is the mechanisms of these enzymes that are the focus of this viewpoint. Whereas alkanes might be boring molecules, we hope to show that the mechanisms by which they are formed in Nature, and about which much still remains to be understood, are definitely not! Although all the various organisms so far investigated appear to synthesize alkanes using fatty aldehydes as the precursor, it has become clear that there are several mechanistically distinct classes of decarbonylases, as illustrated in Figure 1. In insects, the enzyme has been shown to be a cytochrome P450 protein,11,12 whereas in cyanobacteria it is a non-heme di-iron enzyme that is structurally related to enzymes such as ferritin and methane monooxygenase.3,13 In plants, the enzyme is an integral membrane protein that has some sequence similarity to the fatty acid hydroxylase superfamily and stearoyl-CoA desaturase.14 On this basis, it too is presumed to be a metalloenzyme, with iron the most likely metal. The different mechanisms of decarbonylation are reflected in the fate of the aldehyde carbon, which is converted to CO2 in insects,12 HCO2H in cyanobacteria,15,16 and CO in plants and algae.6,17 Figure 1 The biosynthesis of alkanes proceeds in two steps from fatty acyl-CoA esters. The decarbonylation of the intermediate fatty aldehyde proceeds by one of three different mechanisms depending upon the organism. The insect AD is a membrane-bound P450 type ... 2 Insect decarbonylases The biosynthesis of hydrocarbons has been studied in a number of insect species including the domestic house fly, Musca domestica, the fruit fly Drosophila melanogaster, cockroaches Periplaneta americana and Blattella germanica, and the termite Zootermopsis angusticollis.18 Experiments on microsomal preparations of M. domestica demonstrated that molecular oxygen and NADPH were required for the conversion of (Z)-15-tetracosenal to (Z)-9-tricosene and, using 1-14C-material, that the aldehyde carbon was oxidized to CO2 in the process.12 Furthermore, antibodies to either house fly cytochrome P450 reductase or to a cytochrome P450 (CYP6A1) purified from the house fly inhibited (Z)-9-tricosene.19 These initial observations provided support for the involvement of a P450 enzyme in the reaction; this was more recently confirmed with the cloning and heterologous expression of the enzyme, CYP4G1, from Drosophila.11 Further clues to the mechanism of hydrocarbon formation came from analyses of n-tricosane formed by M. domestica microsomal preparations incubated with [2,2-2H2,2-13C]-tetracosanoyl-CoA and [3,3-2H2,3-13C]-tetracosanoyl-CoA which demonstrated that the deuterium atoms on the 2- and 3-positions were retained in the hydrocarbon product.19 Analysis of [l-2H]-tetracosenal incubated with microsomal preparations demonstrated that the aldehyde proton on C-l was transferred to the (Z)-9-tricosene product. Alternative oxidizing agents such as hydrogen peroxide, cumene hydroperoxide, and iodosobenzene were shown to substitute for O2 and NADPH in the reaction. To accommodate these observations, a mechanism has been proposed, shown in Figure 2A, in which the high valent iron-oxo species, resulting from heterolytic cleavage of the O-O bond of the iron-peroxy intermediate, abstracts an electron from the carbonyl group of the aldehyde.19 The reduced iron-oxo species then attacks the carbonyl carbon of the aldehyde to form an iron-hemiacetal diradical. This intermediate is proposed to fragment to form an alkyl radical and an iron-bound formyl radical. In the final step, the alkyl radical then abstracts the formyl hydrogen to produce the hydrocarbon and CO2. Figure 2 Comparison of the deformylation reactions catalyzed by insect AD (CYP4G1) and CYP2B4. A – Deformylation of fatty aldehydes by the insect AD is proposed to start with the high-valent iron-oxo species and results in the formation of CO2. The color-coding ... P450 enzymes catalyze a notably diverse range of oxidative transformations on a very wide range of substrates.20 Even so, the decarbonylase reaction stands out as being unusual. In most cases P450 enzymes oxidize aldehydes to carboxylic acids through a well understood mechanism involving hydrogen atom abstraction by the high valent iron-oxo intermediate followed by “rebound” of the iron-bound hydroxyl group to give the hydroxylated substrate.21 In some cases decarbonylation of the aldehyde does occur, notably in the aromatization reaction of androst-4-ene-3,17-dione to estrone catalyzed by human aromatase and the deformylation of cyclohexanal catalyzed by CYP2B4 (Figure 2B).20 However in these reactions, which are believed to involve the iron-peroxide form of P450, the aldehyde carbon is converted to formate, rather than CO2, and a double bond is introduced into the oxidized product.20 An intriguing question is how CYP4G1 controls the highly reactive iron-oxo intermediate to accomplish oxidative decarbonylation rather than simply oxidizing the aldehyde to a carboxylic acid, which, a priori, would appear to be a more likely fate for the substrate.