The Novel BacterialN-Demethylase PdmAB Is Responsible for the Initial Step ofN,N-Dimethyl-Substituted Phenylurea Herbicide Degradation

Abstract

ABSTRACTThe environmental fate of phenylurea herbicides has received considerable attention in recent decades. The microbial metabolism ofN,N-dimethyl-substituted phenylurea herbicides can generally be initiated by mono-N-demethylation. In this study, the molecular basis for this process was revealed. ThepdmABgenes inSphingobiumsp. strain YBL2 were shown to be responsible for the initial mono-N-demethylation of commonly usedN,N-dimethyl-substituted phenylurea herbicides. PdmAB is the oxygenase component of a bacterial Rieske non-heme iron oxygenase (RO) system. The genespdmAB, encoding the α subunit PdmA and the β subunit PdmB, are organized in a transposable element flanked by two direct repeats of an insertion element resembling ISRh1. Furthermore, this transposable element is highly conserved among phenylurea herbicide-degrading sphingomonads originating from different areas of the world. However, there was no evidence of a gene for an electron carrier (a ferredoxin or a reductase) located in the immediate vicinity ofpdmAB. Without its cognate electron transport components, expression of PdmAB inEscherichia coli,Pseudomonas putida, and other sphingomonads resulted in a functional enzyme. Moreover, coexpression of a putative [3Fe-4S]-type ferredoxin fromSphingomonassp. strain RW1 greatly enhanced the catalytic activity of PdmAB inE. coli. These data suggested that PdmAB has a low specificity for electron transport components and that its optimal ferredoxin may be the [3Fe-4S] type. PdmA exhibited low homology to the α subunits of previously characterized ROs (less than 37% identity) and did not cluster with the RO group involved inO- orN-demethylation reactions, indicating that PdmAB is a distinct bacterial RON-demethylase.