PNNL

Abstract

Acetone carboxylases (ACs) catalyze the metal- and ATP-dependent conversion of acetone and bicarbonate to form acetoacetate, but their preference for either Mn(II) or Fe(II) has been unclear. Enzymes from the proteobacteria Xanthobacter autotrophicus and Aromatoleum aromaticum share 68% primary sequence identity and identical active sites but have been proposed to have different catalytic metals. The two enzymes were expressed in E. coli, dialyzed against chelators, and remetallated with Mn(II) or Fe(II). Electron paramagnetic and Mössbauer spectroscopies identified spectroscopic signatures respectively of Mn(II) or Fe(II) bound at the active site. These experiments showed that the enzymes themselves, without the assistance of chaperones, second metal sites, or post-translational modifications to facilitate correct metal incorporation, and despite the expected thermodynamic preference for Fe(II), each preferred a distinct metal. Moreover, catalysis was likewise associated uniquely with the cognate metal, though either could potentially serve the same Lewis acidic role. We concluded that the protein structure itself serves as a selectivity filter for Mn(II) or Fe(II).