Cloning of a chicken liver cDNA encoding 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase by functional complementation of Escherichia coli pur mutants.
We have used functional complementation of Escherichia coli pur mutants to clone avian cDNA encoding 5-aminoimidazole ribonucleotide (AIR) carboxylase-5-aminoimidazole-4-N-succinocarboxamide ribonucleotide (SAICAR) synthetase, the bifunctional enzyme catalyzing steps 6 and 7 in the pathway for de novo purine nucleotide synthesis. Mutational analyses have been used to establish the structure-function relationship: NH2-SAICAR synthetase-AIR carboxylase-COOH. The amino acid sequence of the SAICAR synthetase domain is homologous to that of bacterial purC-encoded enzymes, and the sequence of the following AIR carboxylase domain is homologous to that of bacterial purE-encoded enzymes. In E. coli, AIR carboxylase is the product of genes purEK with the purK subunit postulated to have a role in CO2 binding. The avian enzyme lacks sequences corresponding to purK yet functions in E. coli. Functional complementation of E. coli pur mutants can be used to clone additional avian cDNAs for de novo purine nucleotide synthesis.
Department of Biochemistry, Purdue University, West Lafayette, IN 47907.
Z D
ZD
Chen
J E
JE
Dixon
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H
Zalkin
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000235:genetics*; D002645:Chickens; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005816:Genetic Complementation Test; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D010453:Peptide Synthases / Q000235:genetics*; D010957:Plasmids; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D012313:RNA; D002262:Carboxy-Lyases; C022378:phosphoribosylaminoimidazole carboxylase; D010453:Peptide Synthases; C022379:phosphoribosylaminoimidazole-succinocarboxamide synthetase