Abstract
Acireductone dioxygenase from Klebsiella oxytoca (KoARD) is an example of the only known metalloenzyme in literature whose chemical pathway diverges depending solely on the identity of the metal ion in its active site. Fe2+-KoARD catalyzes the on-pathway reaction, which recycles methionine in the methionine salvage pathway. Ni2+-KoARD catalyzes the off-pathway reaction, which leads to the formation of 3-(thiomethyl) propionate, formate, and carbon monoxide. The structures of these two proteins have been solved by solution NMR spectroscopy, but due to the paramagnetic nature of the metal center, the active site is modeled using X-ray absorption spectroscopy (XAS). A crystal structure of mouse ARD has also been solved, but is missing the identity of the metal. Crystal structures of Ni-KoARD and Fe-KoARD should provide the structure of the active site and mechanistic information regarding dual chemistry. Pure Ni2+-bound recombinant protein was expressed in minimal media in E. coli by adding a single metal at induction of protein expression. Inductively coupled plasma mass spectrometry showed one mole of nickel per mole of protein. Crystal screening trials of Ni-KoARD yielded needle and plate crystals. X-ray diffraction data was collected on these Ni-KoARD crystals. Purification and crystallization of Fe-KoARD presents a greater challenge due to oxidation of Fe2+ in the active site. XAS of mouse ARD crystals confirmed the identity of the metal in the active site of Ni-MmARD and Co-MmARD crystals. Due to the instability of HsARD expressed in E. coli, a yeast expression system was also developed to allow for eukaryotic post-translational modifications.