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

Abstract

Cyanobacterial aldehyde decarbonylase (cAD) is, structurally, a member of the di-iron carboxylate family of oxygenases. We previously reported that cAD from Prochlorococcus marinus catalyzes the unusual hydrolysis of aldehydes to produce alkanes and formate in a reaction that requires an external reducing system but does not require oxygen (Das et al., 2011, Angew. Chem. 50, 7148–7152). Here we demonstrate that cADs from divergent cyanobacterial classes, including the enzyme from N. puntiformes that was reported to be oxygen dependent, catalyze aldehyde decarbonylation at a much faster rate under anaerobic conditions, and that the oxygen in formate derives from water. The very low activity (< 1 turn-over/h) of cAD appears to result from inhibition by the ferredoxin reducing system used in the assay and the low solubility of the substrate. Replacing ferredoxin with the electron mediator phenazine methosulfate allowed the enzyme to function with various chemical reductants, with NADH giving the highest activity. NADH is not consumed during turn-over, in accord with the proposed catalytic role for the reducing system in the reaction. With octadecanal, a burst phase of product formation, kprod = 3.4 ± 0.5 min−1 is observed indicating that chemistry is not rate-determining under the conditions of the assay. With the more soluble substrate, heptanal, kcat = 0.17 ± 0.01 min−1 and no burst phase is observed, suggesting that a chemical step is limiting in the reaction of this substrate.

Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities

“…S13 G-K). The poor rate of conversion to alkanes was not surprising given the slow turnover (<10 h ) of the decarbonylase enzyme (14).…”

An insect-specific P450 oxidative decarbonylase for cuticular hydrocarbon biosynthesis

“…A bacterial P450 uses a mechanism related to β-hydroxylation to make 1-alkenes (34). The recently described cyanobacterial aldehyde decarbonylases are members of the nonheme diiron oxygenase family (4,7) and function by cleaving a fatty aldehyde with release of formate (5,6,8). Plants appear to use a putative metalloenzyme thought to release CO from the aldehyde (35), although the likely candidate (36) has not been biochemically characterized.…”

“…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)(5)(6)(7)(8) to catalyze the last step ( Fig. 1).…”

Hydrocarbon profiles and phylogenetic analyses of diversified cyanobacterial species

“…Cyanobacterial ADOs are small proteins of 220-250 amino acids, which catalyze the last step of alka(e)ne biosynthesis. These enzymes, which were initially named aldehyde decarbonylase (ADC), given their function of converting Cn fatty aldehydes to formate and the corresponding Cn-1 alka(e)nes [1,19,22], were finally redesignated as aldehyde-deformylating oxygenases (ADOs) because of the oxygenative nature of the reaction they catalyzed [20,21,23]. Dendograms of ADO did not correspond to a classical cyanobacterial phylogeny when compared with the phylogenetic trees of 16S rDNA or AAR (Fig.…”