ABSTRACTThe degradation of the herbicide dicamba is initiated by demethylation to form 3,6-dichlorosalicylate (3,6-DCSA) inRhizorhabdusdicambivoransNdbn-20. In the present study, a 3,6-DCSA degradation-deficient mutant, Ndbn-20m, was screened. A cluster,dsmR1DABCEFGR2, was lost in this mutant. The cluster consisted of nine genes, all of which were apparently induced by 3,6-DCSA. DsmA shared 30 to 36% identity with the monooxygenase components of reported three-component cytochrome P450 systems and formed a monophyletic branch in the phylogenetic tree. DsmB and DsmC were most closely related to the reported [2Fe-2S] ferredoxin and ferredoxin reductase, respectively. The disruption ofdsmAin strain Ndbn-20 resulted in inactive 3,6-DCSA degradation. WhendsmABC, but notdsmAalone, was introduced into mutant Ndbn-20m andSphingobium quisquiliarumDC-2 (which is unable to degrade salicylate and its derivatives), they acquired the ability to hydroxylate 3,6-DCSA. Single-crystal X-ray diffraction demonstrated that the DsmABC-catalyzed hydroxylation occurred at the C-5 position of 3,6-DCSA, generating 3,6-dichlorogentisate (3,6-DCGA). In addition, DsmD shared 51% identity with GtdA (a gentisate and 3,6-DCGA 1,2-dioxygenase) fromSphingomonassp. strain RW5. However, unlike GtdA, the purified DsmD catalyzed the cleavage of gentisate and 3-chlorogentisate but not 6-chlorogentisate or 3,6-DCGAin vitro. Based on the bioinformatic analysis and gene function studies, a possible catabolic pathway of dicamba inR. dicambivoransNdbn-20 was proposed.IMPORTANCEDicamba is widely used to control a variety of broadleaf weeds and is a promising target herbicide for the engineering of herbicide-resistant crops. The catabolism of dicamba has thus received increasing attention. Bacteria mineralize dicamba initially via demethylation, generating 3,6-dichlorosalicylate. However, the catabolism of 3,6-dichlorosalicylate remains unknown. In this study, we cloned a gene cluster,dsmR1DABCEFGR2, involved in 3,6-dichlorosalicylate degradation fromR. dicambivoransNdbn-20, demonstrated that the cytochrome P450 monooxygenase system DsmABC was responsible for the 5-hydroxylation of 3,6-dichlorosalicylate, and proposed a dicamba catabolic pathway. This study provides a basis to elucidate the catabolism of dicamba and has benefits for the ecotoxicological study of dicamba. Furthermore, the hydroxylation of salicylate has been previously reported to be catalyzed by single-component flavoprotein or three-component Rieske non-heme iron oxygenase, whereas DsmABC was the only cytochrome P450 monooxygenase system hydroxylating salicylate and its methyl- or chloro-substituted derivatives.
Role of Cytochrome P450 Monooxygenase in the Bioactivation of Aflatoxin B1