Mechanism and Catalytic Site Atlas

Formyltetrahydrofolate synthetase (FTHFS) is one of the enzymes participating in the transfer of one-carbon units, an essential element of various biosynthetic pathways. FTHFS catalyses the ATP-dependent activation of a formate ion via its addition to the N10 position of tetrahydrofolate. FTHFS is a highly expressed key enzyme in both the Wood-Ljungdahl pathway of autotrophic CO₂ fixation (acetogenesis) and the glycine synthase/reductase pathways of purinolysis. The key physiological role of this enzyme in acetogens is to catalyze the formylation of tetrahydrofolate, an initial step in the reduction of carbon dioxide and other one-carbon precursors to acetate. In purinolytic organisms, the enzymatic reaction is reversed, liberating formate from 10-formyltetrahydrofolate with concurrent production of ATP [PMID:11087401, PMID:10747779]. In many of these processes the transfers of one-carbon units are mediated by the coenzyme tetrahydrofolate (THF). In eukaryotes the FTHFS activity is expressed by a multifunctional enzyme, C-1-tetrahydrofolate synthase (C1-THF synthase), which also catalyses the dehydrogenase and cyclohydrolase activities. Two forms of C1-THF synthases are known [PMID:2836393], one is located in the mitochondrial matrix, while the second one is cytoplasmic. In both forms the FTHFS domain consists of about 600 amino acid residues and is located in the C-terminal section of C1-THF synthase. In prokaryotes FTHFS activity is expressed by a monofunctional homotetrameric enzyme of about 560 amino acid residues [PMID:2200509].

The crystal structure of N(10)-formyltetrahydrofolate synthetase from Moorella thermoacetica shows that the subunit is composed of three domains organised around three mixed beta-sheets. There are two cavities between adjacent domains. One of them was identified as the nucleotide binding site by homology modelling. The large domain contains a seven-stranded beta-sheet surrounded by helices on both sides. The second domain contains a five-stranded beta-sheet with two alpha-helices packed on one side while the other two are a wall of the active site cavity. The third domain contains a four-stranded beta-sheet forming a half-barrel. The concave side is covered by two helices while the convex side is another wall of the large cavity. Arg 97 is likely involved in formyl phosphate binding. The tetrameric molecule is relatively flat with the shape of the letter X, and the active sites are located at the end of the subunits far from the subunit interface [PMID: 10747779].