Saturday, February 18, 2017
Exhibit Hall (Hynes Convention Center)
Aditi Deshpande, Brandeis University, Newton, MA
The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella oxytoca are the only known pair of naturally occurring metalloenzymes with distinct chemical and physical properties determined solely by the identity of the metal (Fe2+ or Ni2+) in the active site. The Fe2+ containing isozyme catalyzes on-pathway chemistry using the substrates acireductone and dioxygen to generate formate and the ketoacid precursor of methionine, whereas the Ni2+ containing isozyme uses the same substrates to catalyze an off-pathway shunt to methylthiopropionate, carbon monoxide and formate. We recently have shown that mammalian homologs of ARD (mouse and human) are also capable of this metal-dependent dual-chemistry in vitro. The possibility of dual chemistry in mammals is interesting since carbon monoxide, which is one of the products of off-pathway reaction, is an anti-apoptotic molecule. In addition, several biochemical and genetic studies have indicated role an inhibitory role of human ARD (HsARD) in cancer. Recombinant mouse and human ARD isozymes were expressed and purified to obtain a homogeneous single transition metal (Mn, Fe, Co, Ni) bound to the enzyme. All the metal-bound proteins exhibited enzymatic activity; but the Fe2+ bound protein showed highest activity than others. Similar to ARD from Klebsiella, both human and mouse ARD bound to Ni2+, Co2+ or Mn2+ exhibited off-pathway chemistry whereas Fe2+-bound protein exhibited on-pathway chemistry. Thermal stability indicated a large difference in melting temperatures of the proteins; with Ni2+ bound mammalian ARD being the most stable followed by Co2+ and Fe2+ and Mn2+ being the least stable. Crystal structures of Ni2+bound mouse ARD co-crystallized with product and substrate analogs provided insight into substrate binding, metal coordination and catalytic mechanism. Solution NMR data of Fe2+, Ni2+ and Co2+ bound-HsARD isozymes indicate that the metal center imparts significant structural differences to the overall protein structure.