Function and structure, subcellular localization and evolution of the encoding gene of pentachlorophenol 4-monooxygenase in sphingomonads

Abstract

Pentachlorophenol 4-monooxygenase (PCP4MO) from Sphingobium chlorophenolicum catalyzes the conversion of pentachlorophenol to tetrachloro-p-hydroquinone in the presence of NADPH and oxygen. It has been shown to be a good point of departure for the study of the detoxification of pollutants by microorganisms and the evolution of the degrading enzymes and their genes. In the present study, we were interested in investigating the catalytic mechanism of active site residues, subcellular localization of PCP4MO in Sphingobium chlorophenolicum ATCC 39723 and the distribution of a pcpB allele in a contaminated ecosystem in Kärkölä, Finland. It was thought these could provide the possibility for the rational engineering of an enzyme for bioremediation, for a better understanding of the general feature of the detoxification of polychlorophenols and for addressing the evolutionary origin of the PCP-degrading gene. We first developed an efficient production and purification system for recombinant PCP4MO by means of which a large quantity of recombinant protein comparable with the native form could be obtained and the investigation of active site residues by site-directed mutagenesis initiated. Secondly, the mutagenesis of three tyrosine residues suggested that Tyr216 in PCP4MO might be critical for the formation of an effective form of PCP to stimulate enzyme reduction or for the maintenance of a favourable conformation for NADPH access to FAD. In addition, the study speculated on the essential role of Tyr234 in stabilizing the NADPH-accessible conformation of FAD. Unexpectedly, contrary to earlier suggestions, Tyr395 made no contribution to PCP binding or PCP hydroxylation. Furthermore, the timing of the translocation and periplasmic localization of PCP4MO in Sphingobium chlorophenolicum ATCC 39723 was revealed by immunochemical and immunocytochemical analyses. The result suggested that the location of a detoxifying enzyme, such as PCP4MO, in the periplasm might be a general feature in the degradation of halogenated xenobiotics to avoid toxic damage to the microorganisms. Finally, the existence of a pcpB allele in the phylogenetically divergent polychlorophenol-degrading sphingomonads in contaminated groundwater in Kärkölä, Finland, has possibly been achieved by a recent horizontal gene transfer.