Abstract
In Neurospora crassa, the mitochondrial arginine biosynthetic enzymes, N-acetylglutamate kinase (AGK) and N-acetyl-gamma-glutamyl-phosphate reductase (AGPR), are generated by processing of a 96-kDa cytosolic polyprotein precursor (pAGK-AGPR). The proximal kinase and distal reductase domains are separated by a short connector region. Substitutions of arginines at positions -2 and -3 upstream of the N terminus of the AGPR domain or replacement of threonine at position +3 in the mature AGPR domain revealed a second processing site at position -20. Substitution of arginine at position -22, in combination with changes at -2 and -3, prevented cleavage of the precursor and identified two proteolytic cleavage sites, Arg-Gly downward arrow Tyr-Leu-Thr at the N terminus of the AGPR domain and Arg-Gly-Tyr downward arrow Ser-Thr located 20 residues upstream. Inhibitors of metal-dependent peptidases blocked proteolytic cleavage at both sites. Amino acid residues required for proteolytic cleavage in the connector were identified, and processing was abolished by mutations changing these residues. The unprocessed AGK-AGPR fusion had both catalytic activities, including feedback inhibition of AGK, and complemented AGK-AGPR- mutants. These resul...Continue Reading
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