Discovery and Characterization of Manganese-dependent Monooxygenases

Project Summary The oxidation of carbon-hydrogen (C-H) bonds is a central biochemical process required by all aerobic organisms. The microbial conversion of these bonds endows potent bioactivity on natural products and enable pathogens to thrive on otherwise inert host-derived biomolecules. These reactions are currently known to be accomplished by a short list of cofactors that include heme, nonheme iron, and copper. While manganese cofactors perform difficult oxidation reactions, including water oxidation within Photosystem II, they are generally not known to be used for C-H bond activation. We recently discovered that the 2-aminoisobutyric acid hydroxylase from Rhodococcus wratislaviensis, AibH1H2, requires manganese to functionalize a strong, aliphatic C-H bond (BDE = 100 kcal/mol). Structural and spectroscopic studies of this enzyme revealed a redox- active, heterobimetallic manganese-iron active site at the locus of O2 activation and substrate coordination. This result expands the known reactivity of biological manganese-iron cofactors, which was previously restricted to single electron transfer or stoichiometric protein oxidation. Since the AibH1H2 cofactor is supported by a protein fold distinct from typical, well-studied bimetallic oxygenases, our proposed research centers on the characterization of ill-defined members of this protein family. Our preliminary results indicate that many of these proteins harbor two active-site metal ions, behave as monooxygenases and, in some cases, display enhanced enzymatic activity when manganese ions are preferentially incorporated within the active site. The core scientific hypothesis for this study is that the unique sequence and structural features of this emergent class of monooxygenases endows embedded manganese ions with otherwise unknown biological reactivity. To investigate this hypothesis, the specific aims of this study are to (1) understand the signature amino acid motifs that differentiate these monooxygenases from their ancestral, structurally-related amidohydrolases, (2) characterize the geometric and electronic structures of mixed iron/manganese-containing enzymatic intermediates, and (3) evaluate the thermodynamic and kinetic competence of cambialistic Mn/Fe monooxygenases. The resulting enzymatic information, methods, and structures will be of specific interest to those in the fields of metallo-enzymology, structural biology, and synthetic bioinorganic chemistry, and of broad interest to microbiologists in general.