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 co-product of alkane synthesis, following the in vitro conversion of octadecanal (R-CHO, where R = n-C17H35) to heptadecane (R-H) by the Nostoc punctiforme AD isolated following its overproduction in Escherichia coli. Instead, we identified formate (HCO2−) as the stoichiometric co-product of the reaction. Results of isotope-tracer experiments indicate that the aldehyde hydrogen is retained in the HCO2− 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 O2-activating non-heme 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.

Cyanobacterial aldehyde decarbonylase (AD) catalyzes conversion of fatty aldehydes (R-CHO) to alka(e)nes (R-H) and formate. Curiously, although this reaction appears to be redox-neutral and formally hydrolytic, AD has a ferritin-like protein architecture and a carboxylate-bridged di-metal cofactor that are both structurally similar to those found in di-iron oxidases and oxygenases. In addition, the in vitro activity of the AD from Nostoc punctiforme (Np) was shown to require a reducing system similar to the systems employed by these O2-utilizing di-iron enzymes. Here, we resolve this conundrum by showing that aldehyde cleavage by the Np AD also requires dioxygen and results in incorporation of 18O from 18O2 into the formate product. AD thus oxygenates, without oxidizing, its substrate. We posit that (i) O2 adds to the reduced cofactor to generate a metal-bound peroxide nucleophile that attacks the substrate carbonyl and initiates a radical scission of the C1-C2 bond, and (ii) the reducing system delivers two electrons during aldehyde cleavage, ensuring a redox-neutral outcome, and two additional electrons to return an oxidized form of the cofactor back to the reduced, O2-reactive form.

Cyanobacterial aldehyde-deformylating oxygenases (ADOs) belong to the ferritin-like diiron-carboxylate superfamily of dioxygen-activating proteins. They catalyze conversion of saturated or mono-unsaturated Cn fatty aldehydes to formate and the corresponding Cn-1 alkanes or alkenes, respectively. This unusual, apparently redox-neutral transformation actually requires four electrons per turnover to reduce the O2 co-substrate to the oxidation state of water and incorporates one O-atom from O2 into the formate co-product. We show here that the complex of the diiron(II/II) form of ADO from Nostoc punctiforme (Np) with an aldehyde substrate reacts with O2 to form a colored intermediate with spectroscopic properties suggestive of a Fe2III/III complex with a bound peroxide. Its Mössbauer spectra reveal that the intermediate possesses an antiferromagnetically (AF) coupled Fe2III/III center with resolved sub-sites. The intermediate is long-lived in the absence of a reducing system, decaying slowly (t1/2 ~ 400 s at 5 °C) to produce a very modest yield of formate (< 0.15 enzyme equivalents), but reacts rapidly with the fully reduced form of 1-methoxy-5-methylphenazine (MeOPMS) to yield product, albeit at only ~ 50% of the maximum theoretical yield (owing to competition from one or more unproductive pathway). The results represent the most definitive evidence to date that ADO can use a diiron cofactor (rather than a homo- or hetero-dinuclear cluster involving another transition metal) and provide support for a mechanism involving attack on the carbonyl of the bound substrate by the reduced O2 moiety to form a Fe2III/III-peroxyhemiacetal complex, which undergoes reductive O-O-bond cleavage, leading to C1–C2 radical fragmentation and formation of the alk(a/e)ne and formate products.

Aldehyde-deformylating oxygenase (ADO) is a ferritin-like nonheme-diiron enzyme that catalyzes the last step in a pathway through which fatty acids are converted into hydrocarbons in cyanobacteria. ADO catalyzes conversion of a fatty aldehyde to the corresponding alk(a/e)ne and formate, consuming four electrons and one molecule of O2 per turnover and incorporating one atom from O2 into the formate coproduct. The source of the reducing equivalents in vivo has not been definitively established, but a cyanobacterial [2Fe-2S] ferredoxin (PetF), reduced by ferredoxin-NADP(+) reductase (FNR) using NADPH, has been implicated. We show that both the diferric form of Nostoc punctiforme ADO and its (putative) diferric-peroxyhemiacetal intermediate are reduced much more rapidly by Synechocystis sp. PCC6803 PetF than by the previously employed chemical reductant, 1-methoxy-5-methylphenazinium methyl sulfate. The yield of formate and alkane per reduced PetF approaches its theoretical upper limit when reduction of the intermediate is carried out in the presence of FNR. Reduction of the intermediate by either system leads to accumulation of a substrate-derived peroxyl radical as a result of off-pathway trapping of the C2-alkyl radical intermediate by excess O2, which consequently diminishes the yield of the hydrocarbon product. A sulfinyl radical located on residue Cys71 also accumulates with short-chain aldehydes. The detection of these radicals under turnover conditions provides the most direct evidence to date for a free-radical mechanism. Additionally, our results expose an inefficiency of the enzyme in processing its radical intermediate, presenting a target for optimization of bioprocesses exploiting this hydrocarbon-production pathway.

Objective: This review summarises the effects of lactic acid bacteria on lactose malabsorption, bacterialaviral or antibiotic associated diarrhoea, and describes the impact of lactic acid bacteria on cancer and the fermentative products in the colon. Results: Eight studies (including 78 patients) demonstrated that lactase de®cient subjects absorbed lactose in yogurt better than lactose in milk, while two studies (25 patients) did not support this. Two studies (22 patients) showed that unfermented acidophilus milk was absorbed better than milk, while six studies (68 patients) found no signi®cant differences. Addition of lactose hydrolysing enzyme, lactase, to milk improved lactose malabsorption in seven studies (131 lactose malabsorbers), while one study (10 malabsorbers) demonstrated no improvement. Lactic acid bacteria alleviated travellers' diarrhoea in one study (94 individuals) while a study including 756 individuals was borderline statistically signi®cant. One study (50 individuals) did not ®nd an effect of lactic acid bacteria on travellers' diarrhoea. Six studies (404 infants) demonstrated a signi®cant effect of lactic acid bacteria on infant diarrhoea, while one study (40 infants) did not. Lactic acid bacteria moderated antibiotic associated diarrhoea in three studies (66 individuals), while two studies (117 individuals) were insigni®cant. Conclusions: Lactase de®cient subjects bene®t from a better lactose absorption after ingestion of yoghurt compared with milk and from milk added lactase, whereas ingestion of unfermented acidophilus milk does not seem to improve lactose absorption. The majority of studies support that lactic acid bacteria alleviate bacterialaviral induced diarrhoea, especially in infants, while the effect on antibiotic associated diarrhoea is less clear.Experimental studies indicate an effect of lactic bacteria on human cell cancer lines, but clinical evidence is lacking. A`stabilising' effect of lactic acid bacteria on the colonic¯ora has not been documented.