Cloning, expression, and characterization of a recombinant bifunctional protein -- porcine AIR carboxylase and SAICAR synthetase

The conversion of aminoimidazole ribonucleotide (AIR) to carboxyaminoimidazole ribonucleotide (CAIR) by phosphoribosylaminoimidazole carboxylase (AIR carboxylase) and the conversion of conversion of ATP, L-aspartate and CAIR to 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide (SAICAR), ADP and phosphate by phosphoribosylaminoimidazolesuccinocarboxamide synthetase (SAICAR synthetase) represent the 7th and 8th steps respectively of de novo purine nucleotide biosynthesis. For the vertebrate enzyme system, AIR carboxylase and SAICAR synthetase are combined in a bifunctional protein encoded by a single gene. Here, a porcine gene encoding AIR carboxylase/SAICAR synthetase was cloned and expressed in Escherichia coli. The native molecular weight of the purified recombinant protein infers an octameric assembly of subunits. Site-directed mutagenesis has identified residues (Lys19, Asp277, and Lys304) critical to AIR carboxylase or SAICAR synthetase activities, indicating a protein defined by separate domains for each catalytic function. The decline in the K m for CAIR in the SAICAR synthetase reaction due to mutations in the AIR carboxylase active site infers some level of active-site coupling in the oligomeric vertebrate enzyme. A double mutant Lys304→Ala/Val 208→Glu exhibits CAIR-dependent ATPase activity in the absence of L-aspartate, suggesting that CAIR binding induces a conformational change in the ATP binding pocket and causes the hydrolysis of ATP or the hydrolysis of the presumed carbonyl phosphate intermediate of CAIR