title: The structure of an enzyme-product complex reveals the critical role of a terminal hydroxide nucleophile in the bacterial phosphotriesterase mechanism.
abstract: A detailed understanding of the catalytic mechanism of enzymes is an important step toward improving their activity for use in biotechnology. In this paper, crystal soaking experiments and X-ray crystallography were used to analyse the mechanism of the Agrobacterium radiobacter phosphotriesterase, OpdA, an enzyme capable of detoxifying a broad range of organophosphate pesticides. The structures of OpdA complexed with ethylene glycol and the product of dimethoate hydrolysis, dimethyl thiophosphate, provide new details of the catalytic mechanism. These structures suggest that the attacking nucleophile is a terminally bound hydroxide, consistent with the catalytic mechanism of other binuclear metallophosphoesterases. In addition, a crystal structure with the potential substrate trimethyl phosphate bound non-productively demonstrates the importance of the active site cavity in orienting the substrate into an approximation of the transition state.
authors.affiliation: Research School of Chemistry, Australian National University, Canberra ACT 0200, Australia.
authors.forename: Colin
authors.initials: C
authors.lastname: Jackson
authors.forename: Hye-Kyung
authors.initials: HK
authors.lastname: Kim
authors.forename: Paul D
authors.initials: PD
authors.lastname: Carr
authors.forename: Jian-Wei
authors.initials: JW
authors.lastname: Liu
authors.forename: David L
authors.initials: DL
authors.lastname: Ollis
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D044345:Phosphoric Triester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012231:Rhizobium / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D044345:Phosphoric Triester Hydrolases