Correction to Oxygen-Independent Alkane Formation by Non-Heme Iron-Dependent Cyanobacterial Aldehyde Decarbonylase: Investigation of Kinetics and Requirement for an External Electron Donor

Abstract

Long chain alkanes are produced by a wide variety of organisms including plants (1–2), insects (3–4) and birds (5), in which they function as water-proofing agents, and by green algae where they serve as a store of cellular energy (6). These alkanes, which are typified by an odd number of carbons, are derived from fatty acid biosynthesis through the elongation of the “standard” C16 and C18 fatty acids by specialized fatty acid synthases to “very long chain” fatty acyl-CoA esters of 20 – 34 carbons (2). These acyl-CoA esters are then reduced to the corresponding aldehydes by fatty acyl-CoA reductase (7). In the final, and chemically most unusual step, cleavage of the bond between the aldehyde carbon and the α-carbon produces an odd-chain alkane; this reaction is catalyzed by aldehyde decarbonylase1 (AD) (8–9). The potential utility of this enzyme has not been lost on those seeking to develop new routes to biofuels (10). It appears that there are three distinct AD enzymes that catalyze different chemical reactions to effect removal of the aldehyde carbon. In higher plants and green algae the enzyme is a metal dependent, integral membrane protein that converts the aldehyde carbon to carbon monoxide (9, 11). In insects AD appears to be a cytochrome p450 enzyme that oxidizes the aldehyde carbon to carbon dioxide (4). In cyanobacteria AD (cAD) was recently found to be a small, soluble protein whose structure, shown in Figure 1, is closely related to the non-heme di-iron oxygenases (10). We previously demonstrated that, in the reaction catalyzed by cAD from the cyanobacterium Prochlorococcus marinus MIT9313, the aldehyde carbon is converted to formate (Figure 1) (12). Labeling studies established that the proton in the alkane derives from water and that the aldehyde proton is retained in formate (12), so that the alkane is formally derived by hydrolysis of the aldehyde, albeit in a very unusual reaction. Similar results have been independently demonstrated by Warui et al. (13) for cAD from a related cyanobacterium, Nostoc punctiformes. Figure 1 A: x-ray structure of cAD from P. marinus MIT9313, illustrating the position of the di-iron center and fatty acid bound at the active site. B: detail of the di-iron center and metal coordinating ligands. C: reaction catalyzed by cAD. The color coding ... Our initial characterization of cAD (12) established that it is an iron-dependent enzyme and that an auxiliary reducing system is required for activity. In this respect, the enzyme is similar to other di-iron oxygenases such as methane mono-oxygenase (14–15). However, we determined that oxygen is not a co-substrate in the reaction; the enzyme is in fact more active under anaerobic conditions. Furthermore, the reducing system appears to be absolutely required for alkane formation even under rigorously anaerobic conditions. EPR experiments provided evidence that free radical species are generated in the mechanism. Based on these results we proposed a tentative mechanism, shown in Figure 2, for the enzyme in which the reducing system plays a catalytic role, possibly serving to generate free radicals to facilitate the chemically difficult cleavage of the aldehyde carbon. Figure 2 Mechanistic proposal for the oxygen-independent formation of alkanes by cAD. In this mechanism the external reducing system functions catalytically to generate a reactive ketyl radical anion and facilitate carbon-carbon bond cleavage. For a detailed discussion ... Our findings stand in contrast to a labeling study by Li et al. (16) using cAD from N. punctiformes, which found that cAD activity was dependent upon the presence of oxygen and demonstrated that oxygen was incorporated into formate. To accommodate this finding the authors proposed a “cryptic oxidation” mechanism in which activated oxygen species react with the aldehyde to facilitate C-C bond cleavage. However, the rate of the oxidative reaction was orders of magnitude slower than the rate we measured for the anaerobic reaction. To address the apparent discrepancies noted above, we have directly compared the activities of cAD from representatives of 4 of the major classes of cyanobacteria: Prochlorococcus marinus MIT9313, Nostoc punctiformes PCC73102, Synechococcus sp. RS9917 and Synechocystis sp. PCC6803. These enzymes each exhibit activity under anaerobic conditions. Focusing on the P. marinus enzyme, for which a structure is available, we have undertaken a detailed investigation of the kinetics of the enzyme and the unusual role of the reducing system in the reaction.