Cloning and structure-function analysis of the chicken glutamine phosphoribosylpyrophosphate amidotransferase

Gaochao Zhou, Purdue University

Abstract

The cDNA for glutamine phosphoribosylpyrophosphate amidotransferase (amidotransferase), the regulatory enzyme of de novo purine nucleotide biosynthesis, has been cloned for the first time from an animal. Previous studies suggest the existence of an Fe or Fe-S component in avian and human amidotransferases. Thus, two modifications are proposed for the chicken amidotransferase maturation. The cloned B. subtilis amidotransferase gene (purF) complements an E. coli purF mutant and overexpresses an active amidotransferase with two normal posttranslational modifications: the cleavage of an 11-amino acid pro-peptide and the assembly of a (4Fe-4S) cluster. However, the chicken cDNA produced an inactive enzyme with an unprocessed pro-peptide in E. coli. Complementation of the E. coli purF mutant was obtained only after the removal of the pro-peptide encoding sequence. The slow growth of the purF mutant with the cDNA encoding chicken amidotransferase lacking the pro-peptide demonstrates that the pro-peptide is not the sole reason for inactivity of the chicken proenzyme and suggests that the Fe component is defective in E. coli. On the other hand, the chicken cDNA encoding the proenzyme was functionally expressed in Ade$\sp-$A, a CHO mutant cell line, while the functional complementation of CHO Ade$\sp-$A cells with B. subtilis purF required a pro-peptide deletion. The slow growth of the CHO Ade$\sp-$A mutant with B. subtilis enzyme not containing the pro-peptide and numerous gene amplifications are evidence for the defect of the (4Fe-4S) cluster of the B. subtilis enzyme in CHO cells. The reciprocal situation of these experiments suggests that the two modifications are required for the chicken amidotransferase to function and that the Fe component in the B. subtilis enzyme must be different from the chicken amidotransferase. Three antibodies for the chicken amidotransferase were made. Using a vaccinia virus-phage T7 expression system, 5 cysteines were examined to determine what role they play in pro-peptide processing. A defective C1F mutant was not processed. Two potential iron ligand cysteines at position 415 and 488 were mutated to serines, resulting in inactive and insoluble enzymes with unprocessed pro-peptides. This suggests that conserved cysteines corresponding to the iron ligands in the B. subtilis enzyme may have similar roles in the avian amidotransferase. A number of structural perturbations adversely affect pro-peptide processing. (Abstract shortened with permission of author.)

Degree

Ph.D.

Advisors

Zalkin, Purdue University.

Subject Area

Biochemistry

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