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.

Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme

“…The metal ion centre also involves the carboxylation of a lysine residue 34 . Both metal ions are seen in the available PTE structures, including engineered mutants 35,36 . However, the ligand-free structure of R18 was found to have a barely occupied b-site, a partially filled a-site and a previously unseen observed g-site ( Supplementary Fig.…”

Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase

“…Wild-type and mutant PTEs were expressed in E. coli BL21-DE3 recAϪ cells grown in TB medium using autoinduction. Purification was performed as described previously (18). SDS/PAGE analysis of pooled active fractions indicated purified PTEs were essentially homogeneous.…”

“…PTEs have a number of attributes that make them interesting in this context. First, the enzymes house a highly reactive binuclear metal ion center that is involved in a relatively simple reaction: namely, a one-step S N 2 displacement reaction for the hydrolysis of the P-O bond of the phosphotriester paraoxon that does not involve any covalent enzyme-substrate intermediates (16)(17)(18). This makes PTE a less complicated system than other enzymes in which conformational changes are involved in multistep reactions and/or reactions involving cofactors.…”

The enzymatic basis for pesticide bioremediation

“…The rapid turnover of OPs is made possible through the use of a binuclear metal active site, which has been characterised crystallographically [60]. Although a wide variety of divalent metal ions can be utilised, the native enzyme contains a heterobinuclear Fe…”

“…-Zn 2+ centre [60]. The catalytic mechanism is thought to proceed via direct in-line nucleophilic attack from a water molecule, activated through its interaction with the Fe 2+ ion, at the electrophilic phosphorus of the substrate, which coordinates to the Zn 2+ ion [61].…”