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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...

Protein L-isoaspartate-(D-aspartate)O-methyltransferases (EC 2.1.1.77), present in a wide variety of prokaryotic and eukaryotic organisms, can initiate the conversion of abnormal L-isoaspartyl residues that arise spontaneously with age to normal L-aspartyl residues. In addition, the mammalian enzyme can recognize spontaneously racemized D-aspartyl residues for conversion to L-aspartyl residues, although no such activity has been seen to date for enzymes from lower animals or prokaryotes. In this work, we characterize the enzyme from the hyperthermophilic archaebacterium Pyrococcus furiosus. Remarkably, this methyltransferase catalyzes both L-isoaspartyl and D-aspartyl methylation reactions in synthetic peptides with affinities that can be significantly higher than those of the human enzyme, previously the most catalytically efficient species known. Analysis of the common features of L-isoaspartyl and D-aspartyl residues suggested that the basic substrate recognition element for this enzyme may be mimicked by an N-terminal succinyl peptide. We tested this hypothesis with a number of synthetic peptides using both the P. furiosus and the human enzyme. We found that peptides devoid of aspartyl residues but containing the N-succinyl group were in fact methyl esterified by both enzymes. The recent structure determined for the Lisoaspartyl methyltransferase from P. furiosus complexed with an L-isoaspartyl peptide supports this mode of methyl-acceptor recognition. The combination of the thermophilicity and the high affinity binding of methylaccepting substrates makes the P. furiosus enzyme useful both as a reagent for detecting isomerized and racemized residues in damaged proteins and for possible human therapeutic use in repairing damaged proteins in extracellular environments where the cytosolic enzyme is not normally found.) is a repair enzyme that catalyzes the S-adenosylmethionine (AdoMet)-dependent 1 methyl esterification of the ␣-carboxyl group of L-isoaspartyl residues that originate from the spontaneous degradation of aspartic acid and asparagine residues in proteins (1-5). The enzyme-mediated methylation reaction is followed by nonenzymatic steps that result in the net conversion of L-isoaspartyl residues to L-aspartyl residues, representing a potentially important mechanism for avoiding the accumulation of damaged proteins as cells age (4 -9). This methyltransferase is found in a wide array of organisms including eubacteria (10), plants (11, 12), nematodes (13), insects (14), and mammals (15). Its amino acid sequence is highly conserved (16). Its functional importance to the bacterium Escherichia coli, the nematode worm Caenorhabditis elegans, and mice has been assessed by analyzing the effect of knockout mutations of its structural genes. In E. coli, methyltransferasedeficient cells are more sensitive to stress in the stationary phase (17), whereas knockout worms show poorer survival in the dauer phase (18). Methyltransferase-deficient mice suffer fatal seizures at an early age (19 -21). Interestingly, the e...

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Crystals of lima been trypsin inhibitor (LBTI) were obtained by using the vapor phase equilibration technique with sodium/potassium tartrate as the precipitating agent. The space group was determined to be cubic, I2(1)3 with a = 110.2. A These crystals diffract to about 1.9 A resolution. Preliminary analysis of self-rotation maps (calculated from native x-ray intensity data) suggests the presence of two monomers in the asymmetric unit. LBTI is very thermostable and retains activity even after boiling for 10 minutes. This property is exploited as part of its purification procedure.