Expression of active domains of a human folate-dependent trifunctional enzyme in Escherichia coli.
The cDNA encoding the human trifunctional enzyme methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase was engineered to contain a prokaryotic ribosome binding site and was expressed under the bacteriophage T7 RNA polymerase promoter in Escherichia coli. Site-directed mutagenesis was used to prepare constructs that encode separately the dehydrogenase/cyclohydrolase (D/C) domain as amino acid residues 1-301, and the synthetase (Syn) domain as residues 304-935. Both domains formed active enzymes thereby demonstrating their ability to fold independently. The full-length enzyme, D/C and Syn domains were expressed at levels 4-, 55- and 3-fold higher than the specific activities found in liver. Additional mutagenesis and independent expression of domains further defined the interdomain region to include amino acids 292-310. The D/C domain was purified to homogeneity by a single affinity chromatographic step, and the full-length protein in a two-step procedure. The kinetic properties of the D/C domain appear unaltered from those of the trifunctional enzyme.
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Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
MacKenzie
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D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005574:Formate-Tetrahydrofolate Ligase / Q000096:biosynthesis* / Q000235:genetics; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; C021836:formyl-methenyl-methylenetetrahydrofolate synthetase; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D000619:Aminohydrolases; D005574:Formate-Tetrahydrofolate Ligase