Atypical protein isoaspartyl residues arise spontaneously during the aging process from the deamidation of protein asparaginyl residues and the isomerization of protein aspartyl residues. These abnormal residues are modified in cells by a strongly conserved protein carboxyl methyltransferase (PCMT) as a first step in a repair pathway. Because a decline in cellular repair mechanisms is hypothesized to contribute to senescence, we determined whether increased PCMT activity was correlated with enhanced longevity. Two ubiquitous promoters were used with the binary GAL4-UAS system to drive PCMT overexpression in Drosophila melanogaster. Flies expressing PCMT activity under the regulation of either the hsp70 or actin5C promoter had enzyme activities that were 3-or 7-fold higher, respectively, than control flies at 29°C. Correlated with the observed increases in PCMT activities, such flies lived on average 32-39% longer than control flies. Lifespan extension was not observed at 25°C with either hsp70-or actin5C-driven expression, indicating a temperature-dependent effect on longevity. We conclude that protein repair is an important factor in the determination of lifespan under certain environmental conditions. PCMT activity may become limiting under mild stress conditions that accelerate rates of protein damage.

Protein L-isoaspartyl methyltransferases (PIMT; EC 2.1.1.77) catalyze the S-adenosylmethionine-dependent methylation of L-isoaspartyl residues that arise spontaneously in proteins with age, thereby initiating a repair process that restores the normal backbone configuration to the damaged polypeptide. In Drosophila melanogaster, overexpression of PIMT in transgenic flies extends the normal life span, suggesting that protein damage can be a limiting factor in longevity. To understand structural features of the Drosophila PIMT (dPIMT) important for catalysis, the crystal structure of dPIMT was determined at a resolution of 2.2 A, and site-directed mutagenesis was used to identify the role of Ser-60 in catalysis. The core structure of dPIMT is similar to the modified nucleotide-binding fold observed in PIMTs from extreme thermophiles and humans. A striking difference of the dPIMT structure is the rotation of the C-terminal residues by 90 degrees relative to the homologous structures. Effectively, this displacement generates a more open conformation that allows greater solvent access to S-adenosylhomocysteine, which is almost completely buried in other PIMT structures. The enzyme may alternate between the open conformation found for dPIMT and the more closed conformations described for other PIMTs during its catalytic cycle, thereby allowing the exchange of substrates and products. Catalysis by dPIMT requires the side chain of the conserved, active site residue Ser-60, since substitution of this residue with Thr, Gln, or Ala reduces or abolishes the methylation of both protein and isoaspartyl peptide substrates.