2 research outputs found
Functional Studies on <i>Oligotropha carboxidovorans</i> Molybdenum–Copper CO Dehydrogenase Produced in <i>Escherichia coli</i>
The
Mo/Cu-dependent CO dehydrogenase (CODH) from <i>Oligotropha
carboxidovorans</i> is an enzyme that is able to catalyze both
the oxidation of CO to CO<sub>2</sub> and the oxidation of H<sub>2</sub> to protons and electrons. Despite the close to atomic resolution
structure (1.1 Ã…), 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 <i>O. carboxidovorans</i> CODH in <i>Escherichia coli</i>. 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 <i>O. carboxidovorans</i>. 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<sub>2</sub> oxidation of Mo/Cu-dependent CODHs in the future
Modulating the Molybdenum Coordination Sphere of <i>Escherichia coli</i> Trimethylamine <i>N</i>‑Oxide Reductase
The
well-studied enterobacterium <i>Escherichia coli</i> present
in the human gut can reduce trimethylamine <i>N</i>-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 <i>in vitro</i> 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 <i>Rhodobacter capsulatus</i> 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