title: Anomalous scattering analysis of Agrobacterium radiobacter phosphotriesterase: the prominent role of iron in the heterobinuclear active site.
abstract: Bacterial phosphotriesterases are binuclear metalloproteins for which the catalytic mechanism has been studied with a variety of techniques, principally using active sites reconstituted in vitro from apoenzymes. Here, atomic absorption spectroscopy and anomalous X-ray scattering 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 (alpha and beta) in the protein expressed in E. coli grown in supplemented medium. Comparison with OpdA expressed in unsupplemented medium demonstrates that the cobalt present in the supplemented medium replaced zinc at the beta-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 these phosphotriesterases are natively heterobinuclear iron-zinc enzymes.
authors: Research School of Chemistry, Building 35, Australian National University, Canberra, ACT 0200, Australia.
authors: Colin J
authors: CJ
authors: Jackson
authors: Paul D
authors: PD
authors: Carr
authors: Hye-Kyung
authors: HK
authors: Kim
authors: Jian-Wei
authors: JW
authors: Liu
authors: Paul
authors: P
authors: Herrald
authors: Natasa
authors: N
authors: Mitić
authors: Gerhard
authors: G
authors: Schenk
authors: Clyde A
authors: CA
authors: Smith
authors: David L
authors: DL
authors: Ollis