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

Abstract

23The herbicide dicamba is initially degrade via demethylation in Rhizorhabdus 24 dicambivorans Ndbn-20. A gene cluster scaffold 66 containing a THF-dependent 25 dicamba methyltransferase dmt and three THF metabolic-related genes, namely, mthfD, 26 dhc and purU, is responsible for dicamba demethylation in this strain. However, the 27 characteristics and functions of MthfD, Dhc and PurU have not been elucidated. In this 28 study, MthfD was synthesized in Escherichia coli BL21(DE3) and purified as a His6-29 tagged protein. Purified MthfD was found to be a monomer, and exhibited 5-CH3-THF 30 dehydrogenase activity in vitro. The Kcat and Km for 5-CH3-THF were 0.23 s -1 and 16.48 31 μM, respectively. However, 5,10-CH2-THF reductase activity was not detected for 32 MthfD yet. Gene disruption results showed that mthfD is essential for dicamba 33 degradation, whereas dhc is dispensable. Our studies revealed that MthfD 34 physiologically is a 5-CH3-THF dehydrogenase that catalyzes the irreversible 35 dehydrogenation of 5-CH3-THF to 5,10-CH2-THF in the THF regeneration pathway 36 during dicamba demethylation in R. dicambivorans Ndbn-20. 37 38 45 This study investigated the characteristics, catalytic activities and function 46 of MthfD. The results revealed that MthfD differs from previously 47 reported MetF in phylogenesis, characteristics and function. MthfD physiologically 48acts as a 5-CH3-THF dehydrogenase but not 5,10-CH2-THF reductase, and it is 49 essential for dicamba catabolism in R. dicambivorans Ndbn-20. This study provides 50 new insights into the mechanism of THF-dependent methyltransferase system. 51 52 53Methyl-substituted compounds such as caffeine, chloromethane, sesamin, vanillate and 54 syringate, are widely present in nature (1-6). And a variety of synthetic methyl-55 substituted aromatics are used in industrial and agricultural production, e.g., dicamba, 56 linuron, chlortoluron, alachlor and isoproturon, which are important herbicides ((7-10). 57Demethylation is usually the initial and critical step in the microbial degradation of 58 methyl-substituted compounds. To date, three types of demethylases from aerobic 59 bacteria have been reported: Rieske non-heme iron oxygenase (RHO) (11, 12), 60 cytochrome P450 (13, 14) and tetrahydrofolate (THF)-dependent methyltransferase (6, 61 15-21). Both RHO and P450 are multi-component monooxygenase systems with 62 NAD(P)H as cofactor, these demethylases add a molecule of oxygen to the methyl 63 group, resulting in removal of the methyl group from the substrate to form 64 formaldehyde. THF-dependent methyltransferase transfers the methyl from the 65 substrates to cofactor THF producing 5-methyltetrahydrofolate (5-CH3-THF), thus its 66 process and mechanism are completely different from RHO-and P450-mediated 67 demethylation. To date, five THF-dependent methyltransferases have been identified: 68 the vanillate methyltransferase LigM and syringate methyltransferase DesA from 69 Sphingomonas paucimobilis SYK-6 (6, 17), the chloromethane methyltransferase 70 CmuAB ...