期刊
BIOCHEMISTRY
卷 51, 期 40, 页码 7908-7916出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi300912n
关键词
-
资金
- National Science Foundation [MCB-1122079]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1122079] Funding Source: National Science Foundation
Cyanobacterial aldehyde decarbonylases (ADs) catalyze the conversion of C-n fatty aldehydes to formate (HCO2-) and the corresponding Cn-1 alk(a/e)nes. Previous studies of the Nostoc punctiforme (Np) AD produced in Escherichia coli (Ec) showed that this apparently hydrolytic reaction is actually a cryptically redox oxygenation process, in which one O-atom is incorporated from O-2 into formate and a protein-based reducing system (NADPH, ferredoxin, and ferredoxin reductase; N/F/FR) provides all four electrons needed for the complete reduction of O-2. Two subsequent publications by Marsh and co-workers [Das, et al. (2011) Angew. Chem. Int. Ed. 50, 7148-7152; Eser, et al. (2011) Biochemistry 50, 10743-10750] reported that their Ec-expressed Np and Prochlorococcus marinus (Pm) AD preparations transform aldehydes to the same products more rapidly by an O-2-independent, truly hydrolytic process, which they suggested proceeded by transient substrate reduction with obligatory participation by the reducing system (they used a chemical system, NADH and phenazine methosulfate; N/PMS). To resolve this discrepancy, we re-examined our preparations of both AD orthologues by a combination of (i) activity assays in the presence and absence of O-2 and (ii) O-18(2) and (H2O)-O-18 isotope-tracer experiments with direct mass-spectrometric detection of the HCO2- product. For multiple combinations of the AD orthologue (Np and Pm), reducing system (protein-based and chemical), and substrate (n-heptanal and n-octadecanal), our preparations strictly require O-2 for activity and do not support detectable hydrolytic formate production, despite having catalytic activities similar to or greater than those reported by Marsh and co-workers. Our results, especially of the O-18-tracer experiments, suggest that the activity observed by Marsh and co-workers could have arisen from contaminating O-2 in their assays. The definitive reaffirmation of the oxygenative nature of the reaction implies that the enzyme, initially designated as aldehyde decarbonylase when the Cl-derived coproduct was thought to be carbon monoxide rather than formate, should be redesignated as aldehyde-deformylating oxygenase (ADO).
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