PubMed · 1718263
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S K Singla. 1991-10-01. Nomenclature errors.. https://doi.org/10.1042/bj2790328
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The purL gene from Lactobacillus casei, encoding phosphoribosylformylglycinamidine synthase II involved in the de novo synthesis of purines, was cloned and sequenced. The putative purL product of 741 amino acids (M(r) of 79,575) shows 25% and 53% identity to the homologous enzymes from Escherichia coli and Bacillus subtilis, respectively. In addition, partial sequences of two other pur genes (purQ and purF) and a possible third gene (purC) were obtained. All these genes are organized in an operon similar to that of B. subtilis. In contrast, the corresponding genes from E. coli and Salmonella typhimurium are scattered through the genome.
The pathway for de novo biosynthesis of purine nucleotides contains two one-carbon transfer reactions catalyzed by glycinamide ribotide (GAR) and 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylases in which N10-formyltetrahydrofolate is the one-carbon donor. We have found that the antifolates methotrexate (MTX) and piritrexim (PTX) completely block the de novo purine pathway in mouse L1210 leukemia cells growing in culture but with only minor accumulations of GAR and AICAR to less than 5% of the polyphosphate derivatives of N-formylglycinamide ribotide (FGAR) which accumulate when the pathway is blocked completely by azaserine. This azaserine-induced accumulation of FGAR polyphosphates is completely abolished by MTX, indicating that inhibition of the pathway is at or before GAR transformylase (reaction 3; Lyons, S. D., and Christopherson, R. I. (1991) Biochem. Int. 24, 187-197). Three h after the addition of MTX (0.1 microM), cellular 5-phosphoribosyl-1-pyrophosphate has accumulated 3.4-fold while 6-methyl-mercaptopurine riboside (25 microM) induces a 6.3-fold accumulation. These data suggest that amido phosphoribosyltransferase catalyzing reaction 1 of the pathway is the primary site of inhibition. In support of this conclusion, we have found that dihydrofolate-Glu5, which accumulates in MTX-treated cells, is a noncompetitive inhibitor of amido phosphoribosyltransferase with a dissociation constant of 3.41 +/- 0.08 microM for interaction with the enzyme-glutamine complex in vitro. Folate-Glu5, MTX-Glu5, PTX, dihydrotriazine benzenesulfonyl fluoride, and AICAR also inhibit amido phosphoribosyltransferase.
Avian glutamine phosphoribosylpyrophosphate amidotransferase contains an NH2-terminal propetide-like sequence. NH2-terminal sequence analysis of immunoaffinity purified enzyme from chicken liver indicates that the propeptide is processed and the mature enzyme starts with Cys. Propeptide processing was investigated by site-directed mutagenesis using a system for expression in HeLa cells. Glutamine-dependent activity and processing were abolished by replacement of the conserved cysteine at position 1, whereas NH3-dependent activity was retained. Cys1 is thus inferred to have a role in glutamine-dependent activity and in propeptide processing. Inactive, insoluble enzymes in which the propeptide was not processed were obtained as a result of replacements of cysteines 415 and 488. Cysteine residues at positions 415 and 488 are inferred to be ligands to an Fe-S cluster on the basis of sequence similarity to the enzyme from Bacillus subtilis. Mutation of Cys269 and Cys295 led to loss of enzyme activity and propeptide processing, although solubility was unchanged. The results suggest that incorporation of an Fe-S cluster is needed for native structure, resultant propeptide processing, and glutamine-dependent activity.