Anomalous scattering analysis of Agrobacterium radiobacter phosphotriesterase: the prominent role of iron in the heterobinuclear active site


Autoria(s): Jackson, Colin J.; Carr, Paul D.; Kim, Hye-Kyung; Liu, Jian-Wei; Herrald, Paul; Mitic, Natasa; Schenk, Gerhard; Smith, Clyde A.; Ollis, David L.
Data(s)

11/05/2006

Resumo

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.

Identificador

http://espace.library.uq.edu.au/view/UQ:79556

Idioma(s)

eng

Publicador

Portland Press Ltd

Palavras-Chave #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 #C1 #250105 Structural Chemistry #250106 Mechanisms of Reactions #270108 Enzymes #780103 Chemical sciences
Tipo

Journal Article