Catalytic Implications from the Drosophila Protein l-Isoaspartyl Methyltransferase Structure and Site-Directed Mutagenesis^,

Abstract

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.

How Oligomerization Contributes to the Thermostability of an Archaeon Protein

“…Crystal structures of PIMT have been reported for both mesophiles and thermophiles (Thermotoga maritima (28), Pyrococcus furiosus (29), humans (30,31), and Drosophila melanogaster (32)), revealing similar three-dimensional structures. The structures include an AdoMet-dependent methyltransferase fold, which is a modified Rossman fold consisting of a central seven-stranded ␤-sheet flanked by ␣-helices on both sides; this structure is common to the entire AdoMet-dependent methyltransferase family, including those having DNA, RNA, proteins, polysaccharides, lipids, and small molecules as substrates for methyltransferase reactions (33).…”

Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning

“…Studies of PIMT from a variety of species have determined that the enzyme works by an ordered sequential mechanism in which it must first bind AdoMet before attaining a conformation allowing protein substrate binding (35,36). Thereafter, PIMT (serendipitously fulfilling one of the important criteria essential for successful phage display) has a low turnover rate for substrate release (37,38).…”

Crystal Structure of the Protein l-Isoaspartyl Methyltransferase from Escherichia coli

“…The biggest difference of these PIMTs' structures is about the conformation of their C-terminuses. The DmPIMT's C-terminal loop is in an ''open conformation'' which lies away from the SAM or SAH binding pocket (the same site) [18], while EcPIMT, HPIMT, and PfPIMT's C-terminal loops lie close upon the SAH binding pocket (Fig. 2d).…”