| Genetic dissection of the p-nitrophenol degradation gene cluster identified in Pseudomonas sp. strain ENV2030 provided insight into the biochemical activity of this operonic system. Each directed transpositional mutation blocked substrate metabolism, supporting the targeted gene's involvement in the pathway. This analysis also suggested polarity in the operonic structure, and further defined the operons. The gene for the first enzyme in the pathway, pnpA, is clustered with two other genes translated in the opposite direction, pnpR and pnpB, while the genes for the enzymes of ring cleavage and subsequent metabolism, pnpC, pnpD and pnpE, are clustered in an apparent ‘lower’ operon.; The putative monooxygenase, PnpA, of this pathway was expressed in both Escherichia coli and Pseudomonas sp. strains, showing conversion of p-nitrophenol to the quinone metabolites, hydroquinone or benzoquinone. The specificity range of the monooxygenase was assayed in E. coli expression experiments and the ENV2030 wildtype strain, showing a very narrow range of acceptable alternate compounds. The putative reductase, pnpB, is believed to be involved with the conversion of benzoquinone to hydroquinone and was expressed in a Pseudomonas strain incapable of degrading p-nitrophenol. Expression of subclones from the upper regulon indicated no need for pnpB, and confirmed the role of pnpR in regulating pnpA.; The lower operon contains the genes for the ring cleavage enzyme PnpC, a putative 1,2-dioxygenase cleavage enzyme, and post cleavage metabolic enzymes PnpD, a dehydrogenase, and PnpE, a maleylacetate reductase, for metabolite entry into central cellular metabolism. Mutagenesis revealed polarity of the operon. Expression of PnpC and putative ferredoxin proteins was assayed in E. coli with gas chromatography/mass spectrometry.; Functional analysis of the p-nitrophenol degrading cluster of ENV2030 through biochemical and genetic means connects the sequences with function. Mutagenesis indicated an operonic structure of ring cleavage genes. Knockout mutagenesis and protein expression assays determined that the genes pnpB and pnpFd are not required for the proposed pathway. The investigation focused on the key enzymes PnpA and PnpC, testing the proposed function suggested from sequence data, and biochemically defined the enzymes within their substrate range. This work validates putative functions suggested in the initial work on the gene cluster. |
