Expression of human NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase in Escherichia coli: purification and partial characterization.
NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is a bifunctional enzyme synthesized as a 37-kDa precursor that is imported into the mitochondria of embryonic and transformed mammalian cells. The cDNA encoding the human bifunctional enzyme was modified to remove nucleotides corresponding to the mitochondrial targeting sequence and was subcloned into a procaryotic expression vector under the control of the T7 RNA polymerase promoter. The soluble dehydrogenase-cyclohydrolase was expressed in Escherichia coli at levels up to 150-fold higher than those found in transformed mammalian cells. Forms of the recombinant enzyme with one, three, or seven additional amino-terminal residues were purified to homogeneity and shown to have similar kinetic properties. Investigation of the absolute requirement of the enzyme for Mg2+ using fluorescence quenching indicates that this ion binds in the absence of substrates.
Yang
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XM
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
MacKenzie
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D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000096:biosynthesis* / Q000302:isolation & purification; D001483:Base Sequence; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000096:biosynthesis* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000302:isolation & purification; D009243:NAD / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; C055342:methylene tetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase; D009243:NAD; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D000619:Aminohydrolases; D008274:Magnesium