Jackson, Colin J. and Carr, Paul D. and Kim, Hye-Kyung and Liu, Jian-Wei and Herrald, Paul and Mitic, Natasa and Schenk, Gerhard and Smith, Clyde A. and Ollis, David L.
Anomalous scattering analysis of Agrobacterium radiobacter
phosphotriesterase: the prominent role of iron in the heterobinuclear
Biochemical Journal, 397.
Bacterial phosphotriesterases are binuclear metalloproteins from which the catalytic
mechanism has been studied with a variety of techniques, principally using active sites
reconstituted in vitro from apo-enzymes. Here, atomic absorption spectroscopy and
anomalous X-ray scattering and have been used to determine the identity of the metals
incorporated into the active site in vivo. We have recombinantly expressed the
phosphotriesterase from Agrobacterium radiobacter (OpdA) in Escherichia coli grown in
medium supplemented with 1 mM CoCl2, and in unsupplemented medium. Anomalous
scattering data, collected from a single crystal at the Fe-K, Co-K and Zn-K edges,
indicate that iron and cobalt are the primary constituents of the two metal binding sites in
the catalytic centre ( and ), in protein expressed in E. coli grown in supplemented
medium. Comparison to OpdA expressed in unsupplemented medium demonstrates that
the cobalt present in the supplemented medium replaced zinc at the -position of the
active site, which results in an increase in the catalytic efficiency of the enzyme. These
results suggest an essential role for iron in the catalytic mechanism of bacterial
phosphotriesterases, and that they are natively heterobinuclear iron-zinc enzymes.
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