Organophosphate hydrolase interacts with ferric-enterobactin and promotes iron uptake in association with TonB-dependent transport system.

Our previous studies have shown the existence of organophosphate hydrolase (OPH) as a part of the inner membrane associated Ton complex (ExbB/ExbD and TonB) of Sphingobium fuliginis. We now show its involvement in iron uptake by establishing direct interactions with ferric-enterobactin. The interactions between OPH and ferric-enterobactin were not affected even when the active site architecture is altered by substituting active site aspartate with either alanine or asparagine. Protein docking studies further substantiated these findings and predicted the existence of ferric-enterobactin binding site that is different from the catalytic site of OPH. A lysine residue (82K) found at the predicted ferric-enterobactin binding site facilitated interactions between OPH and ferric-enterobactin. Substitution of lysine with alanine did not affect triesterase activity, but it abrogated OPH ability to interact with both ferric-enterobactin and ExbD, strengthening further the fact that the catalytic site is not the site for binding of these ligands. In the absence of interactions between OPHK82A and ExbD, OPHK82A failed to target membrane in E. coli cells. The Sphingobium fuliginis TonB-dependent transport (SfTonBDT) system was reconstituted in E. coli GS027 cells generated by deleting the exbD and tonB genes. The E. coli GS030 cells having SfTonBDT system with OPH showed increased iron uptake. Such an increase was not seen in E. coli GS029, cells having SfTonBDT system generated either by omitting OPH or by including its variants, OPHD301A, OPHD301N suggesting a role for OPH in enhanced iron uptake.

Topological analysis of the lipoprotein organophosphate hydrolase from Sphingopyxis wildii reveals a periplasmic localisation.

Organophosphate hydrolase (OPH) is a membrane-associated lipoprotein. It translocates across the inner membrane via the twin-arginine transport pathway and remains anchored to the periplasmic face of the inner membrane through a diacylglycerol moiety linked to the invariant cysteine residue found at the junction of a SpaseII cleavage site. Due to the existence of a transmembrane helix at the C-terminus of the mature OPH, an inner-membrane topology was predicted suggesting the C-terminus of OPH is cytoplasmic. The predicted topology was validated by generating OPH variants either fused in-frame with ß-lactamase or with unique cysteine residues. Sphingopyxis wildii cells expressing OPH variants with Bla fused at the N-terminal, C-terminal or central regions all grew in the presence of ampicillin. Supporting the ß-lactamase reporter assay, the OPH variants having unique cysteine residues at different strategic locations were accessible to the otherwise membrane-impermeant PEG-Mal (methoxypolyethylene glycol maleimide) revealing that, with the exception of the lipoprotein anchor, the entire OPH is in the periplasmic space.

Genome-Guided Insights Reveal Organophosphate-Degrading Brevundimonas diminuta as Sphingopyxis wildii and Define Its Versatile Metabolic Capabilities and Environmental Adaptations.

The complete genome sequence of Brevundimonas diminuta represented a chromosome (∼4.15 Mb) and two plasmids (pCMS1 and pCMS2) with sizes of 65,908 and 30,654 bp, respectively. The sequence of the genome showed no significant similarity with the known bacterial genome sequences, instead showed weak similarity with the members of different genera of family, Sphingomonadaceae. Contradicting existing taxonomic position, the core genome-guided phylogenetic tree placed B. diminuta in the genus Sphingopyxis and showed sufficient genome-to-genome distance warranting a new species name. Reflecting the strains ability to grow in harsh environments, the genome-contained genetic repertoire required for mineralization of several recalcitrant man-made aromatic compounds.

Organophosphate Hydrolase Is a Lipoprotein and Interacts with Pi-specific Transport System to Facilitate Growth of Brevundimonas diminuta Using OP Insecticide as Source of Phosphate.

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.