TY  - JOUR
A1  - Kaufmann, Paul
A1  - Duffus, Benjamin R.
A1  - Teutloff, Christian
A1  - Leimkühler, Silke
T1  - Functional Studies on Oligotropha carboxidovorans Molybdenum-Copper CO Dehydrogenase Produced in Escherichia coli
JF  - Biochemistry
N2  - The Mo/Cu-dependent CO dehydrogenase (CODH) from Oligotropha carboxidovorans is an enzyme that is able to catalyze both the oxidation of CO to CO2 and the oxidation of H-2 to protons and electrons. Despite the close to atomic resolution structure (1.1 angstrom), significant uncertainties have remained with regard to the reaction mechanism of substrate oxidation at the unique Mo/Cu center, as well as the nature of intermediates formed during the catalytic cycle. So far, the investigation of the role of amino acids at the active site was hampered by the lack of a suitable expression system that allowed for detailed site-directed mutagenesis studies at the active site. Here, we report on the establishment of a functional heterologous expression system of O. carboxidovorans CODH in Escherichia coli. We characterize the purified enzyme in detail by a combination of kinetic and spectroscopic studies and show that it was purified in a form with characteristics comparable to those of the native enzyme purified from O. carboxidovorans. With this expression system in hand, we were for the first time able to generate active-site variants of this enzyme. Our work presents the basis for more detailed studies of the reaction mechanism for CO and H-2 oxidation of Mo/Cu-dependent CODHs in the future.
Y1  - 2018
U6  - https://doi.org/10.1021/acs.biochem.8b00128
SN  - 0006-2960
VL  - 57
IS  - 19
SP  - 2889
EP  - 2901
PB  - American Chemical Society
CY  - Washington
ER  - 
TY  - JOUR
A1  - Kaufmann, Hans Paul
A1  - Duffus, Benjamin R.
A1  - Mitrova, Biljana
A1  - Iobbi-Nivol, Chantal
A1  - Teutloff, Christian
A1  - Nimtz, Manfred
A1  - Jaensch, Lothar
A1  - Wollenberger, Ulla
A1  - Leimkühler, Silke
T1  - Modulating the Molybdenum Coordination Sphere of Escherichia coli Trimethylamie N-Oxide Reductase
JF  - Biochemistry
N2  - The well-studied enterobacterium Escherichia coli present in the human gut can reduce trimethylamine N-oxide (TMAO) to trimethylamine during anaerobic respiration. The TMAO reductase TorA is a monomeric, bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor-containing enzyme that belongs to the dimethyl sulfoxide reductase family of molybdoenzymes. We report on a system for the in vitro reconstitution of TorA with molybdenum cofactors (Moco) from different sources. Higher TMAO reductase activities for TorA were obtained when using Moco sources containing a sulfido ligand at the molybdenum atom. For the first time, we were able to isolate functional bis-MGD from Rhodobacter capsulatus formate dehydrogenase (FDH), which remained intact in its isolated state and after insertion into apo-TorA yielded a highly active enzyme. Combined characterizations of the reconstituted TorA enzymes by electron paramagnetic resonance spectroscopy and direct electrochemistry emphasize that TorA activity can be modified by changes in the Mo coordination sphere. The combination of these results together with studies of amino acid exchanges at the active site led us to propose a novel model for binding of the substrate to the molybdenum atom of TorA.
Y1  - 2018
U6  - https://doi.org/10.1021/acs.biochem.7b01108
SN  - 0006-2960
VL  - 57
IS  - 7
SP  - 1130
EP  - 1143
PB  - American Chemical Society
CY  - Washington
ER  -