Purification, characterization, cloning, and amino acid sequence of the bifunctional enzyme 5,10-methylenetetrahydrofolate dehydrogenase/5,10-methenyltetrahydrofolate cyclohydrolase from Escherichia coli.
We have purified the enzyme 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) from Escherichia coli to homogeneity by a newly devised procedure. The enzyme has been purified at least 2,000-fold in a 31% yield. The specific activity of the enzyme obtained is 7.4 times greater than any previous preparation from this source. The purified enzyme is specific for NADP. The protein also contains 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9) activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behavior on a molecular sieving column suggest that the enzyme is a dimer of identical subunits. We have cloned the E. coli gene coding for the enzyme through the use of polymerase chain reaction based on primers designed from the NH2 terminal analysis of the isolated enzyme. We sequenced the gene. The derived amino acid sequence of the enzyme contains 287 amino acids of Mr 31,000. The sequence shows 50% identity to two bifunctional mitochondrial enzymes specific for NAD, and 40-45% identity to the presumed dehydrogenase/cyclohydrolase domains of the trifunctional C1-tetrahydrofolate synthase of yeast mitochondria and cytoplasm and human and rat cytoplasm. An identical sequence of 14 amino acids with no gaps is present in all 7 sequences.
D'Ari
L
L
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Rabinowitz
J C
JC
D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C055342:methylene tetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase; D009243:NAD; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D000619:Aminohydrolases