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J. Biol. Chem., Vol. 280, Issue 26, 24759-24767, July 1, 2005

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A Three-component Dicamba O-Demethylase from Pseudomonas maltophilia, Strain DI-6

GENE ISOLATION, CHARACTERIZATION, AND HETEROLOGOUS EXPRESSION^*

Patricia L. Herman, Mark Behrens, Sarbani Chakraborty, Brenda M. Chrastil, Joseph Barycki, and Donald P. Weeks

From the Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 65888-0664

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 subunits 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.

Received for publication, January 18, 2005 , and in revised form, March 16, 2005.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY786442, AY786443, AY786444, and AY786445.

^* This work was supported by funds from United AgriProducts, Inc. and the Consortium for Plant Biotechnology Research, Inc. This is University of Nebraska Agricultural Research Division journal series number 14702. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68588-0118.

To whom correspondence should be addressed: Dept. of Biochemistry, University of Nebraska-Lincoln, N158 Beadle Center, Lincoln, NE 68588-0664. Tel.: 402-472-7917; Fax: 402-472-7842; E-mail: dweeks{at}unlnotes.unl.edu.

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