Characterization of AMD, the AMP Deaminase Gene in Yeast. Production of amd Strain, Cloning, Nucleotide Sequence, and Properties of the Protein

The structural gene for AMP deaminase (AMD) from Saccharomyces cerevisiae has been cloned and characterized. A yeast strain deficient in AMP deaminase activity was produced and shown to be deficient in AMP deaminase protein by Western blot analysis. The gene for AMP deaminase was located in a λgt11 library of yeast genomic DNA, and a DNA fragment from the λgt11 clone was used to locate homologous DNA in a yeast genomic library in the centromeric plasmid YCp50, a yeast-Escherichia coli shuttle vector. One plasmid was selected for its ability to restore AMP catalytic activity to the deficient strain. Yeast deficient in AMP deaminase or those overproducing the enzyme grow at near normal rates. The open reading frame corresponding to AMD codes for a protein of 810 amino acids, molecular weight 93 286. The yeast AMD transcript is 3.0 ± 0.2 kb, and the transcriptional initiation sites have been identified. Western blot analysis of extracts prepared from actively growing yeast indicates a major band at approximately 96 000 molecular weight with several bands at lower molecular weight, including 83 000. When the AMD gene is expressed in E. coli, the large M_r form of AMP deaminase is produced. These results show that the purified enzyme (M_r = 83 000) is a truncated form of the full-length translation product. No adenine nucleotide binding sites were located based on the consensus sequence from other nucleotide binding proteins. No overall homology was found between yeast AMP deaminase and E. coli AMP nucleosidase. Although their metabolic roles and regulatory mechanisms are similar, these enzymes have arisen from separate ancestral proteins.