A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6:: Purification and characterization

Dicamba O-demethylase is a multicomponent enzyme that catalyzes the conversion of the herbicide 2-methoxy-3,6-dichlorobenzoic acid (dicamba) to 3,6-dichlorosalicylic acid (DCSA). The three components of the enzyme were purified and characterized. Oxygenase(DIC) is a homotrimer (alpha)(3) with a subunit molecular mass of approximately 40 kDa. Ferredoxin(DIC) and reductase(DIC) are monomers with molecular weights of approximately 14 and 45 kDa, respectively. EPR spectroscopic analysis suggested the presence of a single [2Fe-2S]((2+/1+)) cluster in ferredoxin(DIC) and a single Rieske [2Fe-2S]((2+:1+)) cluster within oxygenase(DIC). Consistent with the presence of a Rieske iron-sulfur cluster, oxygenase(DIC) displayed a high reduction potential of E-m.7.0 = -21 rnV whereas ferredoxin(DIC) exhibited a reduction potential of approximately E-m.7.0 = -171 mV. Optimal oxygenase(DIC) activity in vitro depended on the addition of Fe2+. The identification of formaldehyde and DCSA as reaction products demonstrated that dicamba O-demethylase acts as a monooxygenase. Taken together, these data suggest that oxygenase(DIC) is an important new member of the Rieske non-heme iron family of oxygenases. (c) 2005 Elsevier Inc. All rights reserved.