Expression cloning of a human cDNA encoding folylpoly(gamma-glutamate) synthetase and determination of its primary structure.
A human cDNA for folypoly(gamma-glutamate) synthetase [FPGS; tetrahydrofolate:L-glutamate gamma-ligase (ADP forming), EC 6.3.2.17] has been cloned by functional complementation of an Escherichia coli folC mutant. The cDNA encodes a 545-residue protein of M(r) 60,128. The deduced sequence has regions that are highly homologous to peptide sequences obtained from purified pig liver FPGS and shows limited homology to the E. coli and Lactobacillus casei FPGSs. Expression of the cDNA in E. coli results in elevated expression of an enzyme with characteristics of mammalian FPGS. Expression of the cDNA in AUXB1, a mammalian cell lacking FPGS activity, overcomes the cell's requirement for thymidine and purines but does not overcome the cell's glycine auxotrophy, consistent with expression of the protein in the cytosol but not the mitochondria.
Garrow
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Department of Nutritional Sciences, University of California, Berkeley 94720.
Admon
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Shane
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology / Q000235:genetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010453:Peptide Synthases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013552:Swine
D011994:Recombinant Proteins; D004247:DNA; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase