Structural and enzymatic characterization of the phosphotriesterase OPHC2 from Pseudomonas pseudoalcaligenes.
BACKGROUND
Organophosphates (OPs) are neurotoxic compounds for which current methods of elimination are unsatisfactory; thus bio-remediation is considered as a promising alternative. Here we provide the structural and enzymatic characterization of the recently identified enzyme isolated from Pseudomonas pseudoalcaligenes dubbed OPHC2. OPHC2 belongs to the metallo-β-lactamase superfamily and exhibits an unusual thermal resistance and some OP degrading abilities.


RESULTS
The X-ray structure of OPHC2 has been solved at 2.1 Å resolution. The enzyme is roughly globular exhibiting a αβ/βα topology typical of the metallo-β-lactamase superfamily. Several structural determinants, such as an extended dimerization surface and an intramolecular disulfide bridge, common features in thermostable enzymes, are consistent with its high Tm (97.8°C). Additionally, we provide the enzymatic characterization of OPHC2 against a wide range of OPs, esters and lactones.


CONCLUSIONS
OPHC2 possesses a broad substrate activity spectrum, since it hydrolyzes various phosphotriesters, esters, and a lactone. Because of its organophosphorus hydrolase activity, and given its intrinsic thermostability, OPHC2 is an interesting candidate for the development of an OPs bio-decontaminant. Its X-ray structure shed light on its active site, and provides key information for the understanding of the substrate binding mode and catalysis.
URMITE UMR CNRS-IRD 6236, IFR48, Faculté de Médecine et de Pharmacie, Université de la Méditerranée, Marseille, France.
Guillaume
G
Gotthard
Julien
J
Hiblot
Daniel
D
Gonzalez
Mikael
M
Elias
Eric
E
Chabriere
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D044345:Phosphoric Triester Hydrolases / Q000737:chemistry*; D010802:Phylogeny; D017433:Protein Structure, Secondary; D044205:Pseudomonas pseudoalcaligenes / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D044345:Phosphoric Triester Hydrolases