Crystal Structure of Human l-Isoaspartyl Methyltransferase

Abstract

The enzyme L-isoaspartyl methyltransferase initiates the repair of damaged proteins by recognizing and methylating isomerized and racemized aspartyl residues in aging proteins. The crystal structure of the human enzyme containing a bound S-adenosyl-L-homocysteine cofactor is reported here at a resolution of 2.1 Å. A comparison of the human enzyme to homologs from two other species reveals several significant differences among otherwise similar structures. In all three structures, we find that three conserved charged residues are buried in the protein interior near the active site. Electrostatics calculations suggest that these buried charges might make significant contributions to the energetics of binding the charged S-adenosyl-L-methionine cofactor and to catalysis. We suggest a possible structural explanation for the observed differences in reactivity toward the structurally similar L-isoaspartyl and D-aspartyl residues in the human, archael, and eubacterial enzymes. Finally, the human structure reveals that the known genetic polymorphism at residue 119 (Val/Ile) maps to an exposed region away from the active site.The loss of biological functions in the aging process can be attributed in part to nonenzymatic chemical reactions that degrade biomolecules including DNA, proteins, and small molecules. We have been interested in enzymatic mechanisms from which organisms have evolved to limit the accumulation of isomerized and racemized aspartyl residues, which form spontaneously from normal L-aspartic acid and L-asparagine residues within proteins as they age. Much attention has been directed at understanding the action of the L-isoaspartate(Daspartate) O-methyltransferase or protein L-isoaspartyl methyltransferase (PIMT) 1 (EC 2.1.1.77). PIMT is an enzyme that recognizes altered aspartyl residues in proteins and polypeptides and drives their eventual conversion to normal L-aspartyl residues (1-3). In the best-characterized pathway, a methyl group is transferred from S-adenosyl-L-methionine (AdoMet) to form a methyl ester on the ␣-carboxyl group of an L-isoaspartyl residue. Subsequent nonenzymatic reactions result in rapid L-succinimide formation and hydrolysis, which generates some "repaired" L-aspartyl residues as well as some L-isoaspartyl residues, which can then enter the cycle again for eventual conversion to the normal peptide linkage. PIMTs have been highly conserved during evolution and are found in almost all eucaryotic cells as well as in most archaebacteria and most Gram-negative eubacteria (4, 5).Recently, three-dimensional structures have been determined for PIMT from the hyperthermophilic eubacterium Thermotoga maritima (6) and the thermophilic archaebacterium Pyrococcus furiosus (7). With the exception of a C-terminal domain apparently unique to the T. maritima enzyme, these enzymes have similar amino acid sequences and a similar three-dimensional fold (5). For the P. furiosus enzyme, the crystal structures of AdoMet-liganded and isoaspartyl peptideliganded forms have revealed how the enzyme can re...

Structure of Protein Phosphatase Methyltransferase 1 (PPM1), a Leucine Carboxyl Methyltransferase Involved in the Regulation of Protein Phosphatase 2A Activity

“…4B). A similar surrounding for AdoMet was observed in the protein L-isoaspartyl-methyltransferase (PIMT) that also catalyzes methyl group transfer to a charged carboxylate group (26). Because AdoMet contains both a charged sulfonium atom and peptidic group, charged side chains will contribute favorably to binding.…”

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).…”

Protein Isoaspartate Methyltransferase Is a Multicopy Suppressor of Protein Aggregation in Escherichia coli

“…In the best-characterized pathway, a methyl group is transferred from S-adenosyl-L-methionine to form a methyl ester on the ␣-carboxyl group of an isoaspartyl residue. Subsequent nonenzymatic reactions result in the rapid formation of an unstable succinimide intermediate which can yield a normal L-aspartyl residue upon hydrolysis, thus ultimately catalyzing net repair of the damaged site (25). The formation of isoaspartyl residues can result in heterogeneity or loss of protein function.…”