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  - Mitrova, Biljana
A1  - Tadjoung Waffo, Armel Franklin
A1  - Kaufmann, Paul
A1  - Iobbi-Nivol, Chantal
A1  - Leimkühler, Silke
A1  - Wollenberger, Ulla
T1  - Trimethylamine N-Oxide Electrochemical Biosensor with a Chimeric Enzyme
JF  - ChemElectroChem
N2  - For the first time, an enzyme-based electrochemical biosensor system for determination of trimethylamine N-oxide (TMAO) is described. It employs an active chimeric variant of TorA in combination with an enzymatically deoxygenating system and a low-potential mediator for effective regeneration of the enzyme and cathodic current generation. TMAO reductase (TorA) is a molybdoenzyme found in marine and most enterobacteria that specifically catalyzes the reduction of TMAO to trimethylamine (TMA). The chimeric TorA, named TorA-FDH, corresponds to the apoform of TorA from Escherichia coli reconstituted with the molybdenum cofactor from formate dehydrogenase (FDH). Each enzyme, TorA and TorA-FDH, was immobilized on the surface of a carbon electrode and protected with a dialysis membrane. The biosensor operates at an applied potential of -0.8V [vs. Ag/AgCl (1M KCl)] under ambient air conditions thanks to an additional enzymatic O-2-scavenger system. A comparison between the two enzymatic sensors revealed a much higher sensitivity for the biosensor with immobilized TorA-FDH. This biosensor exhibits a sensitivity of 14.16nA/M TMAO in a useful measuring range of 2-110M with a detection limit of LOD=2.96nM (S/N=3), and was similar for TMAO in buffer and in spiked serum samples. With a response time of 16 +/- 2 s, the biosensor is stable over prolonged daily measurements (n=20). This electrochemical biosensor provides suitable applications in detecting TMAO levels in human serum.
KW  - trimethylamine N-oxide (TMAO)
KW  - TMAO reductase
KW  - chimeric enzyme
KW  - molybdoenzyme
KW  - electrochemical biosensor
Y1  - 2018
U6  - https://doi.org/10.1002/celc.201801422
SN  - 2196-0216
VL  - 6
IS  - 6
SP  - 1732
EP  - 1737
PB  - Wiley-VCH
CY  - Weinheim
ER  -