The roles of the gentisate 1,2-dioxygenases DsmD and GtdA in the catabolism of the herbicide dicamba in Rhizorhabdus dicambivorans Ndbn-20.

3-Chlorogentisate is a key intermediate in the catabolism of the herbicide dicamba in R. dicambivorans Ndbn-20. In this study, we identified two gentisate 1,2-dioxygenases (GDOs), DsmD and GtdA, from Ndbn-20. The amino acid sequence similarity between DsmD and GtdA is 51%. Both of them are dimers and showed activities to gentisate and 3-chlorogentisate but not 3,6-dichlorogentisate (3,6-DCGA) or 6-chlorogentisate in vitro. The k(cat)/K-m of DsmD for 3-chlorogentisate was 28.7 times higher than that of GtdA, whereas the k(cat)/K-m of DsmD for gentisate was only one-fourth of that of GtdA. Transcription of dsmD was dramatically induced by 3-chlorogentisate but not gentisate, whereas gtdA was not induced. Disruption of dsmD resulted in a significant decline in the degradation rates of 3-chlorogentisate and dicamba but had no effect on the degradation of gentisate, whereas the result of disruption of gtdA was converse; the disruption of both dsmD and gtdA led to the inability to degrade 3-chlorogentisate and gentisate. This study revealed that 3-chlorogentisate but not gentisate or 3,6-DCGA is the ring-cleavage substrate in the dicamba degradation pathway in R. dicambivorans Ndbn-20; DsmD is specifically responsible for cleavage of 3-chlorogentisate, whereas GtdA is a general GDO involved in the catabolism of various natural aromatic compounds.