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Anomalous scattering analysis of Agrobacterium radiobacter phosphotriesterase: the prominent role of iron in the heterobinuclear active site
Jackson, Colin J.; Carr, Paul D.; Kim, Hye-Kyung; Liu, Jian-Wei; Herrald, Paul; Mitic, Natasa; Schenk, Gerhard; Smith, Clyde A.; Ollis, David L.
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.
Keyword(s): Chemistry; Agrobacterium radiobacter; anomalous scattering; heterobinuclear; iron–zinc; metallophosphoesterase; phosphotriesterase
Publication Date:
2006
Type: Journal article
Peer-Reviewed: Yes
Institution: Maynooth University
Citation(s): 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. (2006) Anomalous scattering analysis of Agrobacterium radiobacter phosphotriesterase: the prominent role of iron in the heterobinuclear active site. Biochemical Journal, 397. pp. 501-508. ISSN 0264-6021
Publisher(s): Portland Press
File Format(s): application/pdf
Related Link(s): http://mural.maynoothuniversity.ie/3666/1/GS_Anomalous_Scattering_2.pdf
First Indexed: 2020-01-31 06:03:35 Last Updated: 2020-04-02 07:27:38