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Projekt
Anoxic Enzymatic Conversion of Acetylene
Antragsteller
Professor Dr. Oliver Einsle
Fachliche Zuordnung
Stoffwechselphysiologie, Biochemie und Genetik der Mikroorganismen
Förderung
Förderung von 2011 bis 2015
Projektkennung
Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 210678598
The gas acetylene (ethyne) undergoes two known enzymatic conversions in nature. Nitrogenase, the enzyme required for the reductive fixation of dinitrogen into bioavailable ammonium, reduces acetylene in a two-electron step to ethylene (ethene). In contrast, the tungsten / [4Fe:4S] enzyme acetylene hydratase catalyzes the hydratation of the gas to yield acetaldehyde. The proteins are well characterized structurally and functionally, but the mode and exact site of acetylene binding and the mechanism of its conversion remain under heavy debate for both cases. Clearly, the mechanisms of acetylene conversion follow entirely different routes: while nitrogenase most likely binds acetylene (and other substrates) to the [Mo:7Fe:9S:X]:homocitrate FeMo cofactor, from which electrons of a low redox potential get transferred directly to the substrate, the tungstoprotein acetylene hydratase has a distinct, binding pocket pre-formed by a ring of hydrophobic amino acids. Here the reaction may not involve the formation of a metal-carbon bond, but rather the activation of a water molecule coordinated to tungsten that subsequently attacks the triple bond of the alkyne. We will establish and optimize both native and recombinant production of acetylene hydratase in our laboratory, create point-specific mutants that will be analyzed for enzymatic activity and obtain high-resolution structures of wild type and variant proteins pressurized with acetylene or other candidate ligands. Nitrogenase will be isolated from Azotobacter vinelandii and assayed for acetylene reduction activity. Acetylene, and in particular the strong ligand CO will be used in pressurization experiments to obtain atomic resolution data from which we expect to gain essential mechanistic clues.
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