Detection of formate, rather than carbon monoxide, as the stoichiometric coproduct in conversion of fatty aldehydes to alkanes by a cyanobacterial aldehyde decarbonylase

Abstract

The second of two reactions in a recently discovered pathway through which saturated fatty acids are converted to alkanes (and unsaturated fatty acids to alkenes) in cyanobacteria entails scission of the C1-C2 bond of a fatty aldehyde intermediate by the enzyme aldehyde decarbonylase (AD), a ferritin-like protein with a dinuclear metal cofactor of unknown composition. We tested for and failed to detect carbon monoxide (CO), the proposed C1-derived coproduct of alkane synthesis, following the in vitro conversion of octadecanal (R-CHO, where R = n-C(17)H(35)) to heptadecane (R-H) by the Nostoc punctiforme AD isolated following its overproduction in Escherichia coli. Instead, we identified formate (HCO(2)(-)) as the stoichiometric coproduct of the reaction. Results of isotope-tracer experiments indicate that the aldehyde hydrogen is retained in the HCO(2)(-) and the hydrogen in the nascent methyl group of the alkane originates, at least in part, from solvent. With these characteristics, the reaction appears to be formally hydrolytic, but the improbability of a hydrolytic mechanism having the primary carbanion as the leaving group, the structural similarity of the ADs to other O(2)-activating nonheme di-iron proteins, and the dependence of in vitro AD activity on the presence of a reducing system implicate some type of redox mechanism. Two possible resolutions to this conundrum are suggested.

Figure 1

Reconstructed mass spectra showing formate production in reactions of Np AD. Reactions were carried out at 22 °C for 3 h. The panels show spectra generated by integration of the formyl-2NPH peaks from single-anion chromatograms for the reactions with (A) natural-abundance R-CHO, (B) R-¹³CHO and (C) R-¹³C²HO. With the exception of the gray bars in A, all reactions had 50 µM Np AD and 0.50 mM substrate. The gray bars in A are from a control reaction lacking Np AD and containing 0.50 mM of each of the three substrates. In B, the red bars are from the reaction in natural-abundance H₂O and the blue bars are from the reaction in 78% ²H₂O.

Scheme 1

Three possible outcomes of the Np AD reaction. (A) Reaction proposed by Schirmer, et al.; (B) simplest (non-redox) reaction suggested by the observation of formate production in this study; (C) alternative, cryptically redox conversion suggested by the requirement for the reducing system and the structural similarity of the ADs to O₂-activating di-iron proteins.