Molecules to Burn: A Mechanistic Characterization of Cyanobacterial Aldehyde Deformylating Oxygenase.

[摘要] The development of hydrocarbon based biofuels that can replace fossil fuels is essential to address the challenge of energy sustainability. However, there are few known biosynthetic pathways to produce these molecules and, generally, they are not well understood. The focus of this dissertation is to explore one of the very few biosynthetic routes to produce entirely unfunctionalized hydrocarbons through investigation of the highly unusual reaction catalyzed by cyanobacterial aldehyde deformylating oxygenase (cADO).To investigate the proton transfer step, solvent isotope effect (SIE) studies were undertaken. No appreciable difference in rate in D2O or H2O was observed, implying that proton transfer is not a kinetically significant step. However, when the ratio of protium to deuterium in the product alkane was measured as a function of the mole fraction of D2O, a D2OSIEobs of 2.19 ± 0.02 was observed. We interpret this SIE as most likely arising from a reactant state equilibrium isotope effect on a proton donor with an inverse fractionation factor, for which Φ = 0.45, consistent with an iron-bound water molecule being the proton donor to the alkane.Substrate analogs and binding channel mutations were used to investigate substrate binding or product release acting as a non-chemical rate limiting step. The kinetics of the mutants were investigated using octadecanal and, although no increase apparent rate was observed, two mutants displayed shifts in KM. These results suggest the hydrophobic pocket may be important in determining the binding affinity of long chain substrates. Protein film voltammetry experiments were used to explore the electrochemistry of cADO. The midpoint reduction potential was determined to be -73 ± 10 mV (vs SHE). Catalytic cyclic voltammetry with heptanal indicated a lower limit on alkane turnover of kobs > 0.63 per s, significantly faster than the rate of ~1 per min observed in solution. Interestingly, an alternative reaction was observed with enzyme and O2 indicating a futile cycle leading to H2O2 formation. These observations indicate that inefficient interactions with the reducing system or a partitioning effect between alkane and H2O2 turnover may be responsible for the sluggish activity of cADO.

[发布机构] University of Michigan

[效力级别] Non-Heme Iron Oxygenase

[关键词] Biofuels;Non-Heme Iron Oxygenase;Aldehyde Decarbonylase;Protein Film Voltammetry;Solvent Isotope Effects;Enzyme Kinetics;Biological Chemistry;Science;Chemical Biology