Abstract

The herbicide dicamba is initially degrade via demethylation in Rhizorhabdus dicambivorans Ndbn-20. A gene cluster scaffold 66 containing a THF-dependent dicamba methyltransferase dmt and three THF metabolic-related genes, namely, mthfD, dhc and purU, is responsible for dicamba demethylation in this strain. However, the characteristics and functions of MthfD, Dhc and PurU have not been elucidated. In this study, MthfD was synthesized in Escherichia coli BL21(DE3) and purified as a His6-tagged protein. Purified MthfD was found to be a monomer, and exhibited 5-CH3-THF dehydrogenase activity in vitro. The Kcat and Km for 5-CH3-THF were 0.23 s-1 and 16.48 M, respectively. However, 5,10-CH2-THF reductase activity was not detected for MthfD yet. Gene disruption results showed that mthfD is essential for dicamba degradation, whereas dhc is dispensable. Our studies revealed that MthfD physiologically is a 5-CH3-THF dehydrogenase that catalyzes the irreversible dehydrogenation of 5-CH3-THF to 5,10-CH2-THF in the THF regeneration pathway during dicamba demethylation in R. dicambivorans Ndbn-20.