Abstract
Organophosphate hydrolase (OPH), encoded by the organophosphate degradation (opd) island, hydrolyzes the triester bond found in a variety of organophosphate insecticides and nerve agents. OPH is targeted to the inner membrane ofBrevundimonas diminutain a pre-folded conformation by thetwinargininetransport (Tat) pathway. 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. 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. Substitution of the conserved lipobox cysteine to serine resulted in release of OPH into the periplasm, confirming that OPH is a lipoprotein. Analysis of purified OPH revealed that it was modified with the fatty acids palmitate and stearate. 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. Interaction of OPH with PstS appears to facilitate transport of Pigenerated from organophosphates due to the combined action of OPH and periplasmically located phosphatases. Consistent with this model,opdnull mutants ofB. diminutafailed to grow using the organophosphate insecticide methyl parathion as sole source of phosphate.
Highlights
Organophosphate hydrolase (OPH) associates with cell membranes and membrane-associated OPH has been purified from a number of sources (3, 9 –13)
In this study we have investigated the interaction of the organophosphate hydrolase, OPH, from B. diminuta, with the cytoplasmic membrane and with other cellular proteins
OPH contains an alanine residue at the ϩ2 position and according to the sorting rules for E. coli lipoproteins [42] should be translocated to the outer membrane by the Lol machinery
Summary
B. diminuta cultures were grown either in LB medium or in HEPES minimal medium. HEPES minimal medium was predase;DDM,n-dodecyl-D-maltoside;Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; IMAC, immobilized-metal affinity chromatography; BN-PAGE, blue native-PAGE.
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