Novel Three-Component Rieske Non-Heme Iron Oxygenase System Catalyzing the N -Dealkylation of Chloroacetanilide Herbicides in Sphingomonads DC-6 and DC-2

Abstract

ABSTRACT Sphingomonads DC-6 and DC-2 degrade the chloroacetanilide herbicides alachlor, acetochlor, and butachlor via N -dealkylation. In this study, we report a three-component Rieske non-heme iron oxygenase (RHO) system catalyzing the N -dealkylation of these herbicides. The oxygenase component gene cndA is located in a transposable element that is highly conserved in the two strains. CndA shares 24 to 42% amino acid sequence identities with the oxygenase components of some RHOs that catalyze N - or O -demethylation. Two putative [2Fe-2S] ferredoxin genes and one glutathione reductase (GR)-type reductase gene were retrieved from the genome of each strain. These genes were not located in the immediate vicinity of cndA . The four ferredoxins share 64 to 72% amino acid sequence identities to the ferredoxin component of dicamba O -demethylase (DMO), and the two reductases share 62 to 65% amino acid sequence identities to the reductase component of DMO. cndA , the four ferredoxin genes, and the two reductases genes were expressed in Escherichia coli , and the recombinant proteins were purified using Ni-affinity chromatography. The individual components or the components in pairs displayed no activity; the enzyme mixture showed N -dealkylase activities toward alachlor, acetochlor, and butachlor only when CndA-His 6 was combined with one of the four ferredoxins and one of the two reductases, suggesting that the enzyme consists of three components, a homo-oligomer oxygenase, a [2Fe-2S] ferredoxin, and a GR-type reductase, and CndA has a low specificity for the electron transport component (ETC). The N -dealkylase utilizes NADH, but not NADPH, as the electron donor.