The environmental fate of phenylurea herbicides has received considerable attentions in recent decades.Microbial metabolism of N-N-dimethyl-substituted phenylurea herbicides can generally be initiated by N-demethylation.However, the molecular basis is still unknown.Here, we described a Rieske non-heme iron oxygenase (RO) gene from a phenylurea herbicides degrading strain Sphingobium sp.YBL2, pudmAB, is responsible for the initial N-demethylation of commonly used N-N-dimethyl-substituted phenylurea herbicides.pudmAB, encoding the α subunit PudmA, and β subunit PudmB.Unfortunately, there was no evidence for a gene coding for a ferredoxin or a reductase in the immediate vicinity ofpudmAB.However, expression of PudmAB in Escherichia coli, Pseudomonas and other sphingomonads, without its cognate electron transport components, resulted in a functional enzyme.Furthermore, co-expression of a putative [3Fe-4S]-type ferredoxin from Sphingomonas sp.RW1 remarkably enhance the catalytic activity of PudmAB in E.coli, suggesting PudmAB has low specificity for electron transport components.PudmA exhibited low homology to the α subunits of characterized ROs (less than 36% identity) and formed a new branch in the phylogenetic tree.Moreover, PudmA did not cluster with the ROs group involved in O-or N-demethylation reactions, indicating PudmAB is a distinct bacterial RO N demethylase.