Cloning and characterization of mammaliancDNAs encoding adenylosuccinate lyase and GAR synthetase-AIR synthetase-GAR transformylase

Junko Aimi, Purdue University

Abstract

The pathway for de novo purine nucleotide biosynthesis is conserved among all organisms that have been studied thus far. However, previous studies have indicated that the structural organization and regulation of the pathway vary from species to species. To investigate the molecular genetics of this pathway in higher eukaryotes, we have developed a strategy to clone a number of mammalian cDNAs encoding enzymes responsible for de novo purine nucleotide biosynthesis. We have utilized functional complementation of Eschericia coli pur gene mutants with an avian liver cDNA expression library to isolate cDNAs encoding adenylosuccinate lyase (ASL) as well as the trifunctional enzyme, glycinamide ribonucleotide synthetase (GARS)-aminoimidazole ribonucleotide synthetase (AIRS)-glycinamide ribonucleotide transformylase (GART). The avian cDNA encoding GARS-AIRS-GART was obtained by functional complementation of an E. coli purM mutant strain. The human cDNA of the trifunctional enzyme was subsequently isolated from a human liver cDNA library using the avian cDNA as a probe. DNA sequence analyses demonstrated that the human and avian clones both encode a single polyprotein which exhibits a very high homology to the bacterial purD, purM, and purN encoded proteins. Studies have also revealed that avian and human livers express an additional mRNA which encodes only GARS. The deduced amino acid sequence of the avian ASL cDNA revealed extensive homology to the Bactillus subtilis purB gene product which catalyzes two steps in the de novo purine biosynthetic pathway. Further comparison analysis also displayed regions of amino acid sequence identities with the human urea cycle enzyme, argininosuccinate lyase, as well as two additional enzymes, E. coli fumarase and aspartase. These enzymes each catalyze similar $\beta$-elimination reactions which generate fumarate as a product. The conserved amino acids are, therefore, likely to represent residues which may be important for structure or catalysis. ASL deficiencies have been noted in two siblings diagnosed with autism and severe psychomotor retardation. Additional evidences have suggested that they may have an altered form of the enzyme. To identify possible mutations responsible for the reduced activity and heat lability of the enzyme, the corresponding human ASL cDNA was isolated by screening a human liver library. Subsequently, total RNA from cultured lymphoblasts of the autistic children was used to generate overlapping cDNA fragments for ASL using the polymerase chain reaction (PCR). Sequence analysis revealed a single nucleotide substitution (T $\to$ C) which predicts a conversion of amino acid residue 413 from serine to proline. Additional studies are now being carried out to assess the consequences of this point mutation on the activity of ASL. This is the first report of a molecular defect associated with infantile autism.

Degree

Ph.D.

Advisors

Dixon, Purdue University.

Subject Area

Biochemistry|Molecular biology

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