Purification, characterization, cloning, and amino acid sequence of the bifunctional enzyme 5,10-methylenetetrahydrofolate dehydrogenase/5,10-methenyltetrahydrofolate cyclohydrolase from Escherichia coli.

The 3-D structure of a folate-dependent dehydrogenase/cyclohydrolase bifunctional enzyme at 1.5 å resolution

“…An important clue comes from the analysis of sequences from the D/C family. Previous alignments and analyses revealed a pattern of conserved residues, concentrated in two distinct regions: 78-102 and 272-280 (Figure 2a) [23][24][25]. Following the latest publication of a sequence alignment for D/C enzymes [26], we have mapped to the protein surface the residues that are conserved in 90% of the sequences from enzymes of this family (Figure 3).…”

Impact of Mutating the Key Residues of a Bifunctional 5,10-Methylenetetrahydrofolate Dehydrogenase-Cyclohydrolase from Escherichia coli on Its Activities

“…24 Subsequently, with a 2000-fold purified enzyme, specific activities of 200 μmol min −1 mg −1 for dehydrogenase and 33 μmol min −1 mg −1 for cyclohydrolase (∼16.5% of dehydrogenase activity) were reported. 1 However, the study used different buffers for the dehydrogenase and cyclohydrolase assays. In our study, we used the E. coli ΔfolD/ p-fhs strain to purify FolD and its mutants to ensure no contaminating presence of the host FolD to determine K m , V max , k cat , and k cat /K m (Table 3).…”

“…Thus, in our study, the dehydrogenase activity is more similar to that in the first report 24 and the cyclohydrolase activity to that in the second report. 1 With the exception of the FolD from a purine-requiring organism, Leishmania, 3 FolD proteins from other organisms, e.g., human (cytoplasmic or mitochondrial), A. baumannii, Photobacterium phosphoreum, yeast, porcine, and rabbit liver, 19,22,37−40 possess approximately equal or higher specific activities for the cyclohydrolase activity than for the dehydrogenase activity. FolD structures have revealed the presence of N-and Cterminal domains of the α/β fold connected by two long helices 4,[15][16][17]19,20 and identified the conserved motifs, YX 3 K and S-Q-P, important for substrate binding and catalysis.…”

“…Therefore, WT FolD and other mutants were purified using a His tag, and used in the assays. 1 Briefly, the dehydrogenase and cyclohydrolase activities were assayed in a buffer consisting of 0.1 M potassium maleate (pH 7.6) and 10 mM β-mercaptoethanol. The kinetic constants of FolD (WT and mutants) with respect to their dehydrogenase activity were determined for 5,10-CH 2 -THF and NADP + .…”

“…16,19 Furthermore, significant differences in the kinetic properties of EcoFolD were reported in the two independent studies. 1,24 Although E. coli serves as an important bacterial model, structure−function analysis of EcoFolD has not been studied in any detail. Previously, in a genetic screen, 25 we isolated E. coli strains (A48 and B22), which allowed initiation with a mutant initiator tRNA.…”