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. 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 : 501-508.


Author Jackson, Colin J.
Carr, Paul D.
Kim, Hye-Kyung
Liu, Jian-Wei
Herrald, Paul
Mitic, Natasa
Schenk, Gerhard
Smith, Clyde A.
Ollis, David L.
Title Anomalous scattering analysis of Agrobacterium radiobacter phosphotriesterase: the prominent role of iron in the heterobinuclear active site
Journal name Biochemical Journal   Check publisher's open access policy
ISSN 0264-6021
Publication date 2006-05-11
Sub-type Article (original research)
DOI 10.1042/BJ20060276
Volume 397
Start page 501
End page 508
Total pages 8
Place of publication London
Publisher Portland Press Ltd
Collection year 2006
Language eng
Subject C1
250105 Structural Chemistry
250106 Mechanisms of Reactions
270108 Enzymes
780103 Chemical sciences
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 P) 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.
Keyword Agrobacterium Radiobacter
Anomalous Scattering
Heterobinuclear
Iron-zinc
Metallophosphoesterase
Phosphotriesterase
Biochemistry & Molecular Biology
Purple Acid-phosphatase
Binuclear Metal Center
Pseudomonas-diminuta
Bacterial Phosphotriesterase
Parathion Hydrolase
Directed Evolution
Catalytic-activity
Escherichia-coli
Enzyme
Expression
Q-Index Code C1
Additional Notes DOI 10.1042/BJ20060276

 
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