title: A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6: gene isolation, characterization, and heterologous expression.
abstract: Dicamba O-demethylase is a multicomponent enzyme from Pseudomonas maltophilia, strain DI-6, that catalyzes the conversion of the widely used herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid) to DCSA (3,6-dichlorosalicylic acid). We recently described the biochemical characteristics of the three components of this enzyme (i.e. reductase(DIC), ferredoxin(DIC), and oxygenase(DIC)) and classified the oxygenase component of dicamba O-demethylase as a member of the Rieske non-heme iron family of oxygenases. In the current study, we used N-terminal and internal amino acid sequence information from the purified proteins to clone the genes that encode dicamba O-demethylase. Two reductase genes (ddmA1 and ddmA2) with predicted amino acid sequences of 408 and 409 residues were identified. The open reading frames encode 43.7- and 43.9-kDa proteins that are 99.3% identical to each other and homologous to members of the FAD-dependent pyridine nucleotide reductase family. The ferredoxin coding sequence (ddmB) specifies an 11.4-kDa protein composed of 105 residues with similarity to the adrenodoxin family of [2Fe-2S] bacterial ferredoxins. The oxygenase gene (ddmC) encodes a 37.3-kDa protein composed of 339 amino acids that is homologous to members of the Phthalate family of Rieske non-heme iron oxygenases that function as monooxygenases. Southern analysis localized the oxygenase gene to a megaplasmid in cells of P. maltophilia. Mixtures of the three highly purified recombinant dicamba O-demethylase components overexpressed in Escherichia coli converted dicamba to DCSA with an efficiency similar to that of the native enzyme, suggesting that all of the components required for optimal enzymatic activity have been identified. Computer modeling suggests that oxygenase(DIC) has strong similarities with the core alphasubunits of naphthalene 1,2-dioxygenase. Nonetheless, the present studies point to dicamba O-demethylase as an enzyme system with its own unique combination of characteristics.
authors.affiliation: Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 65888-0664, USA.
authors.forename: Patricia L
authors.initials: PL
authors.lastname: Herman
authors.forename: Mark
authors.initials: M
authors.lastname: Behrens
authors.forename: Sarbani
authors.initials: S
authors.lastname: Chakraborty
authors.forename: Brenda M
authors.initials: BM
authors.lastname: Chrastil
authors.forename: Joseph
authors.initials: J
authors.lastname: Barycki
authors.forename: Donald P
authors.initials: DP
authors.lastname: Weeks
mesh_terms: D000325:Adrenodoxin / Q000737:chemistry; D000595:Amino Acid Sequence; D015139:Blotting, Southern; D002723:Chlorobenzoates; D003001:Cloning, Molecular; D003470:Culture Media; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D003996:Dicamba / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry; D015723:Gene Library; D007501:Iron / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000378:metabolism; D010090:Oxidoreductases, O-Demethylating / Q000737:chemistry* / Q000235:genetics*; D010100:Oxygen / Q000737:chemistry; D010105:Oxygenases / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry; D012459:Salicylates / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
chemical_list: D002723:Chlorobenzoates; D003470:Culture Media; D017931:DNA Primers; D005288:Ferredoxins; D011994:Recombinant Proteins; D012459:Salicylates; D000325:Adrenodoxin; C487853:3,6-dichlorosalicylic acid; D004247:DNA; D007501:Iron; D010088:Oxidoreductases; D010090:Oxidoreductases, O-Demethylating; C502811:dicamba O-demethylase; D010105:Oxygenases; D010100:Oxygen; D003996:Dicamba