Cloning of Two Gene Clusters Involved in the Catabolism of 2,4-Dinitrophenol by Paraburkholderia sp. Strain KU-46 and Characterization of the Initial DnpAB Enzymes and a Two-Component Monooxygenase DnpC1C2

Abstract

Besides an industrial pollutant, 2,4-dinitrophenol (DNP) has been used illegally as a weight loss drug that had claimed human lives. Little is known about the metabolism of DNP, particularly among Gram-negative bacteria. In this study, two non-contiguous genetic loci of Paraburkholderia (formerly Burkholderia) sp. strain KU-46 genome were identified and four key initial genes (dnpA, dnpB, and dnpC1C2) were characterized to provide molecular and biochemical evidence for the degradation of DNP via the formation of 4-nitrophenol (NP), a pathway that is unique among DNP utilizing bacteria. Reverse transcription PCR analysis indicated that the dnpA gene encoding the initial hydride transferase (28 kDa), and the dnpB gene encoding a nitrite-eliminating enzyme (33 kDa), are inducible by DNP and the two genes are organized in an operon. Purified DnpA and DnpB from overexpression clones in Escherichia coli effected the transformation of DNP to NP via the formation of hydride-Meisenheimer complex of DNP. The function of DnpB appears new since all homologs of DnpB sequences in the protein database are annotated as putative nitrate ABC transporter substrate-binding proteins. The gene cluster responsible for the degradation of DNP after NP formation was designated dnpC1C2DXFER. DnpC1 and DnpC2 were functionally characterized as the respective FAD reductase and oxygenase components of the two-component NP monooxygenase. Both NP and 4-nitrocatechol were shown to be substrates, producing hydroquinone and hydroxyquinol, respectively. Elucidation of the hqdA1A2BCD gene cluster allows the delineation of the final degradation pathway of hydroquinone to {beta}-ketoadipate prior to its entry to the tricarboxylic acid cycle.\n\nImportanceThis study fills a gap in our knowledge and understanding of the genetic basis and biochemical pathway for the degradation of 2,4-dinitrophenol (DNP) in Gram-negative bacteria, represented by the prototypical Paraburkholderia sp. strain KU-46 that metabolizes DNP through the formation of 4-nitrophenol, a pathway unseen by other DNP utilizers. The newly cloned genes could serve as DNA probes in biomonitoring as well as finding application in new biocatalyst development to access green chemicals. By and large, knowledge of the diverse strategies used by microorganisms to degrade DNP will contribute to the development of bioremediation solutions since DNP is an industrial pollutant used widely in the chemical industry for the synthesis of pesticides, insecticides, sulfur dyes, wood preservatives, and explosives, etc. (119 words)