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  -