The properties of 5-methyltetrahydrofolate dehydrogenase (MthfD) and its role in the tetrahydrofolate (THF)-dependent dicamba demethylation system inRhizorhabdus dicambivoransNdbn-20

Abstract

ABSTRACTThe herbicide dicamba is initially degrade via demethylation inRhizorhabdus dicambivoransNdbn-20. A gene cluster scaffold 66 containing a THF-dependent dicamba methyltransferasedmtand three THF metabolic-related genes, namely,mthfD,dhcandpurU, 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 inEscherichia coliBL21(DE3) and purified as a His6-tagged protein. Purified MthfD was found to be a monomer, and exhibited 5-CH3-THF dehydrogenase activityin vitro. TheKcatandKmfor 5-CH3-THF were 0.23 s−1and 16.48 μM, respectively. However, 5,10-CH2-THF reductase activity was not detected for MthfD yet. Gene disruption results showed thatmthfDis essential for dicamba degradation, whereasdhcis 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 inR. dicambivoransNdbn-20.