| dc.description.abstract | Phosphonates are a class of organophosphorus compounds with a characteristic carbon–phosphorus bond. The carbon–phosphorus lyase complex, encoded by the genes phnGHIJKLM, catalyzes the cleavage of the stable carbon–phosphorus bond of organophosphonates to the corresponding hydrocarbon and inorganic phosphate. Recently, complexes of this enzyme containing five subunits (PhnG-H-I-J-K), four subunits (PhnG-H-I-J), and two subunits (PhnG-I) were purified after expression in Escherichia coli.
Here we used mass spectrometry, ultracentrifugation, chemical cross-linking, N-terminal protein sequencing and single-particle cryo-electron microscopy experiments, revealing subunits interactions and construct structural models for the PhnG2H2I2J2 and PhnG2H2I2J2K C–P lyase complexes from E. coli. The proposed stoichiometry, subunit interaction and structural model of PhnG2H2I2J2 complex agreed quite well with the crystal structure that was published later. PhnG2H2I2J2 is a two-fold symmetric hetero-octamer. Its two PhnJ subunits provide two identical binding sites for PhnK. Only one PhnK binds to PhnG2H2I2J2 due to steric hindrance. Moreover, binding of PhnK exposes the active site residue, Gly32 of PhnJ, located near the interface between PhnJ and PhnH. This structural information provides a basis for further deciphering of the reaction mechanism of the C-P lyase.
A group of organophosphorus compounds, the phosphotriester plasticizers and flame retardants, has recently become widely used. This group of compounds lacks an easily hydrolyzable bond, rendering them inert to typical phosphotriesterases. A phosphotriesterase from Sphingobium sp. strain TCM1 (Sb-PTE) has recently been reported to catalyze the hydrolysis of organophosphorus flame retardants. This enzyme was expressed in Escherichia coli, and the activity with a wide variety of organophosphorus substrates has been characterized. Sb-PTE exhibits catalytic activity against flame retardants, plasticizers, and industrial solvents.
In this study, we use mass spectrometry and peptide mapping experiments, revealing the active site residues and reaction mechanism for Sb-PTE. The proposed active site residues of Sb-PTE complex agreed quite well with the crystal structure that was published later. The enzyme catalyzes the hydrolysis of substrates by activation of a nucleophilic water molecule for direct attack at the phosphorus center. | en |
