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Primers used for PCR amplification and site-directed mutagenesis are listed in Table 2. Strains and plasmids used in the present work are shown in Table 1. In this report we demonstrate that OPH is a lipoprotein and that it plays an essential role in the acquisition of phosphate from OP insecticides. However, the mechanism by which OPH is anchored to the inner membrane and the physiological role of OPH are currently unclear. Proteinase K treatment confirmed that OPH is exported to the periplasmic side of the inner membrane in Brevundimonas diminuta and dependence on the Tat pathway was demonstrated because substitution of the invariant arginine residues of the Tat signal peptide affected both processing and localization of OPH ( 15). Twin-arginine signal peptides serve to target proteins to the twin-arginine protein transport (Tat) pathway, which translocates folded proteins across the bacterial cytoplasmic membrane ( 14). Analysis of the amino acid sequences of OPH proteins indicates that all of them contain a predicted signal peptide harboring a well defined twin-arginine (Tat) motif. OPH associates with cell membranes and membrane-associated OPH has been purified from a number of sources ( 3, 9, – 13). Structural analysis shows that OPH contains a TIM barrel-fold as seen in most of the members of amidohydrolase superfamily proteins ( 8). Considering its catalytic efficiency and broad substrate range, it has been assumed that OPH has evolved to degrade organophosphate (OP) insecticides accumulated in agricultural soils ( 7). Although its physiological substrate is unknown, OPH hydrolyzes paraoxon at a rate approaching the diffusion limit ( k cat/ K m 10 8 m −1 s −1) ( 6). Due to the mobile nature of the opd island, identical opd genes are found among bacterial strains isolated from different geographical regions ( 4, 5). OPH is encoded by the opd ( organo phosphate degrading) gene found on dissimilar plasmids and the opd gene has recently been shown to be a part of an integrative mobilizable element (IME) ( 4). The 39-kDa monomer requires Zn + ions as cofactor ( 3). Membrane-associated organophosphate hydrolase (OPH) 3 hydrolyzes the triester bond found in a variety of organophosphate insecticides and nerve agents ( 1, 2).
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diminuta failed to grow using the organophosphate insecticide methyl parathion as sole source of phosphate. Consistent with this model, opd null mutants of B. Interaction of OPH with PstS appears to facilitate transport of P i generated from organophosphates due to the combined action of OPH and periplasmically located phosphatases. Membrane-bound OPH was shown to interact with the outer membrane efflux protein TolC and with PstS, the periplasmic component of the ABC transporter complex (PstSACB) involved in phosphate transport. Analysis of purified OPH revealed that it was modified with the fatty acids palmitate and stearate. Substitution of the conserved lipobox cysteine to serine resulted in release of OPH into the periplasm, confirming that OPH is a lipoprotein. Treatment of cells producing native OPH with the signal peptidase II inhibitor globomycin resulted in accumulation of most of the pre-OPH in the cytoplasm with negligible processed OPH detected in the membrane. The OPH signal peptide contains an invariant cysteine residue at the junction of the signal peptidase (Spase) cleavage site along with a well conserved lipobox motif.
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OPH is targeted to the inner membrane of Brevundimonas diminuta in a pre-folded conformation by the twin arginine transport (Tat) pathway. Organophosphate hydrolase (OPH), encoded by the organophosphate degradation ( opd) island, hydrolyzes the triester bond found in a variety of organophosphate insecticides and nerve agents.