Most organisms possess bifunctional FolD [5,10-methylenetetrahydrofolate (5,10-CH2-THF) dehydrogenase-cyclohydrolase] to generate NADPH and 1 0-formyltetrahdrofolate (10-CHO-THF) required in various metabolic steps. In addition, some organisms including Clostridium perfringens possess another protein, Fhs (formyltetrahydrofolate synthetase), to synthesize 10-CHO-THF. Here, we show that unlike the bifunctional FolD of Escherichia coli (Eco FolD), and contrary to its annotated bifunctional nature, C. perfringens FolD (CpeFolD) is a monofunctional 5,10-CH2-THF dehydrogenase. The dehydrogenase activity of Cpe FolD is about five times more efficient than that of Eco FolD. The 5,10-methenyltetrahydrofolate (5,1 0-CH+ -THF) cyclohydrolase activity in C. perfringens is provided by another protein, FchA (5,10-CH+ -THF cyclohydrolase), whose cyclohydrolase activity is ~10 times more efficient than that of Eco FolD. Kinetic parameters for Cpe Fhs were also determined for utilization of all of its substrates. Both Cpe FolD and Cpe FchA are required to substitute for the single bifunctional FolD in E. coli. The simultaneous presence of Cpe FolD and Cpe FchA is also necessary to rescue an E. colifolD deletion strain (harbouring Cpe Fhs support) for its formate and glycine auxotrophies, and to alleviate its susceptibility to trimethoprim (an antifolate drug) or UV light. The presence of the three clostridial proteins (FolD, FchA and Fhs) is required to maintain folate homeostasis in the cell.
ASJC Scopus subject areas