The inability of E. coli expressing OpdA to grow using paraoxon as a sole phosphorus source indicates that one of the reasons why it cannot grow on DMP is its inability to hydrolyze DMP to methyl phosphate under phosphate-limiting conditions. Based on this finding, we sought to isolate and characterize the phosphohydrolase gene from E. aerogenes to test if coexpressing it and OpdA would allow E. coli to grow with paraoxon as the sole phosphorus source.
The phosphohydrolase gene was selected for its ability to enable E. coli
to use DMP as a sole phosphorus source. It was found that the gene was part of an operon homologous to the G3P uptake operon of E. coli
One function of the ugp
operon is to recycle phospholipid metabolites like G3P and glycerophosphoryl diesters (5
). A second possible function is to regulate cAMP levels in the cell. Expression of the E. coli ugp
operon is induced under both phosphate- and carbon-limiting conditions from separate promoters (22
). As the E. aerogenes ugp
operon has all of the genes and is induced under phosphate-limiting conditions in a similar way to the E. coli ugp
operon, it is likely that the operons have similar functions. The lack of a cAMP receptor protein binding site in the promoter region suggests that the E. aerogenes ugp
operon is not expressed under carbon-limiting conditions and has no direct role in regulating cAMP levels, although this idea was not tested further. Although GpdQ displays no sequence similarity to UgpQ, considering the operon in which it is located and the high activity displayed towards a probable natural substrate (GPE), it is likely that the function of the two phosphodiesterases is similar.
While the components of the two operons are similar, the gene arrangement within them differs. In E. coli
, the gene order is ugpBACEQ
, whereas in E. aerogenes
, the order is ugpAEQCB
(Fig. ). In E. coli
, the operon is transcribed monocistronically from either of the two promoters (38
). We have not investigated the regulation of the E. aerogenes gpdQ
operon. The inability of a GpdQ+
strain to grow on DMP and its ability to grow on demeton suggest that a functional UgpB is required for growth on DMP. As the function of E. coli
UgpB is to bind G3P and glycerophosphoryl diesters to enable their transport into the cytoplasm via the UgpACE complex, it is likely that this lack of growth is due to an inability of the truncated form of UgpB to bind DMP (40
). The inhibition of GpdQ+
cell growth on either DMP or demeton by paraoxon and the inhibition of GpdQ+
cell growth on demeton by paraoxon are consistent with the notion that paraoxon is a competing substrate for the growth-rate limiting hydrolysis of demeton and/or DMP. These facts also suggest that the site of inhibition is at the point of hydrolysis, not transport.
It is of interest to note that the amino acid sequence of UgpB from E. aerogenes is homologous to that of the putative sugar binding protein isolated on the same fragment as PdeA from D. acidovorans (expect value, 6e − 11). The proximity of a UgpB homologue and a phosphodiesterase in D. acidovorans suggests that PdeA is also part of a ugp-like operon. Unfortunately, it is unknown if these genes are part of a larger operon or if they are expressed under phosphate-limiting conditions.
The results from the in vitro assays of purified GpdQ on various substrates help explain the growth assay results. GpdQ activity towards demeton is evidence that the growth of GpdQ+ cells is due to hydrolysis of demeton to DMP by GpdQ, not by an E. coli enzyme. The low in vitro activity towards paraoxon indicates the most likely point of GpdQ+ growth inhibition on DMP, i.e., a competing substrate. The relatively high specific activity towards demeton suggests that the faster removal of demeton compared to paraoxon allows time for the subsequent hydrolysis of DMP. The reasons why GpdQ has higher activity towards the phosphorothiolate demeton than towards paraoxon remain unknown. The higher activity of GpdQ towards paraoxon at high pH levels in contrast to other tested substrates is intriguing and awaits further examination. Also of interest are the contrasting pH optima of the phosphodiesterase activities of GpdQ and PdeA from D. acidovorans. Considering the amino acid homology between these two enzymes, this difference is interesting and requires further investigation.
The proteolytic activation of GpdQ is reminiscent of that reported for PAP and may be significant considering their structural similarity. Recombinant human PAP displays increased activity after proteolytic cleavage and removal by trypsin of a tripeptide segment adjacent to the active site (15
). Size exclusion chromatography (data not shown) indicates that GpdQ remains intact, suggesting limited proteolysis of loop regions in the protein analogous to that seen in PAP. It has been proposed that this cleavage regulates PAP activity by raising kcat
and shifting the pH optimum. Considering that GpdQ synthesis is induced under phosphate-limiting conditions, it remains unclear why its activity might be further regulated by proteolysis.
Comparison of the previously determined amino acid composition of GpdQ to a conceptual translation of gpdQ
indicates that the two are most likely the same protein (17
). Sequence analysis and structure prediction show that GpdQ is a member of the recently identified family of class III 3′,5′-cyclic nucleotide phosphodiesterases (39
) and, more broadly, the family of metallophosphoesterases identified by Koonin (28
). The conserved amino acids of the phosphoesterase motif are involved in coordination of the binuclear metal center at the active site and metal-bound water nucleophiles in other members of this metallophosphoesterase family (26
). Accordingly, it is likely that two metal ions are coordinated at the catalytic center of GpdQ. PdeA is activated by Mg2+
, but the effect of other divalent metal ions was not reported (44
). Previous characterization of pH suggested that three Zn2+
and three Mn2+
ions were bound per hexamer, although Gerlt and Wan reported difficulties in determining the metal ion concentration with certainty (16
). Considering the similarity of GpdQ to binuclear metallophosphatases and the lack of relationship with mononuclear metallophosphatases, it is probable that GpdQ has two metal ions per monomer, possibly one Zn2+
and one Mn2+
per monomer. The predicted secondary and tertiary structure of GpdQ, namely a double-β sandwich surrounded on both sides by α helices and characterized by the βαβαβ motif, is similar to that of other binuclear metallophosphatases such as PAP (43
) and the Ser/Thr protein phosphatases (25
The inhibition of growth of a GpdQ+ strain on DMP by paraoxon suggests that under certain conditions organophosphate pesticides can act as antibiotics. The term antibiotics is used here in a general sense to describe a compound that can inhibit the growth of an organism under particular conditions, rather than in the usual and more specific sense to describe bacterial growth inhibitors in a medical environment. The ability of OpdA to enable growth in the presence of paraoxon raises the possibility that the role of phosphotriesterases in bacteria may be more than simply the generation of phosphate sources from organophosphate pesticides. In effect, phosphotriesterases can act as antibiotic resistance enzymes. While it is true that, in the long term, paraoxon would be broken down to provide a phosphorus source, in the short term, the generation of a phosphorus source from paraoxon is of secondary importance compared to the removal of paraoxon as a growth inhibitor. The establishment of a phosphotriesterase via gene transfer or gene duplication and divergence from GpdQ would provide a growth advantage under such conditions.
The reason(s) why DH10B is unable to use DMP as a sole phosphorus source remain uncertain. Assuming that DH10B has a functional ugp
operon, our results suggest at least two reasons. On the one hand, the inability of GpdQ−
cells to grow with paraoxon as the sole phosphorus source suggests that E. coli
does not express an enzyme capable of hydrolyzing DMP under phosphate-limiting conditions. On the other hand, the inability of GpdQ+
cells to grow on DMP lends weight to the argument that E. coli
lacks the capacity to take up DMP. It also remains unclear what enzyme in the GpdQ+
strain hydrolyzes methyl phosphate to phosphate. Given the phosphomonoesterase activity of GpdQ, it is possible that GpdQ catalyzes this step in addition to the phosphodiesterase step. However, an E. coli
phosphomonoesterase is more likely responsible. Alkaline phosphatase turns over methyl phosphate readily (36
). However, as this enzyme is expressed in the periplasm, the issue of methyl phosphate transport from the cytoplasm to the periplasm is raised. Little has been reported about this issue.
Despite the similarity of the E. aerogenes ugp
operon to the E. coli ugp
operon, its in vivo function remains unclear. The similarity suggests that the functions of the two operons are similar, i.e., the catabolism of phospholipids. However, the high activity of GpdQ towards the phosphorothiolate pesticide demeton clouds the issue. As the amino acid sequence of the previously described phosphohydrolase was not reported, it is uncertain if the two proteins are identical. If the two enzymes are different in sequence and activity, then the demeton activity may be due to evolutionary changes in response to pesticides in the environment. If the two enzymes are the same, then the demeton activity may be a remarkable example of catalytic promiscuity (37
The growth of strain DH10B coexpressing GpdQ and OpdA when paraoxon is the sole phosphate source shows that E. coli can be used for selection of OpdA mutants with the ability to hydrolyze paraoxon. In addition, the GpdQ+ strain might also be useful for the bioprospecting of new phosphotriesterases from wild-type organisms that produce DMP from phosphotriesters, especially in cases where the other product is not colored or fluorescent. The use of this system for selecting OpdA mutants with higher activity towards poor substrates appears promising and is under further investigation.