Protein Repair Methyltransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Abstract

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...

Crystal Structure of Human l-Isoaspartyl Methyltransferase

“…This structural complex provides a framework for understanding the substrate specificities of the enzymes from various sources. L-IsoAsp residues can be methylated by PIMT from all three organisms, whereas the D-Asp amino acid can be methylated by PIMT from human and P. furiosus (8) but not from T. maritima (5). PIMT from human and P. furiosus both have a proline in a key position (Pro-49/Pro-65) at which PIMT from T. maritima is replaced by a valine (Val-45).…”

“…However, despite their sequence similarities, the kinetic properties and substrate specificities of the enzymes from various species are distinct. For example, the T. maritima enzyme is unable to methylate polypeptides containing D-aspartyl residues (5), whereas the P. furiosus enzyme is able to recognize such racemized residues (8).…”

Differential proteomic analysis of developmental stages of Acca sellowiana somatic embryos

“…Protein spot 23, only found in the torpedo stage somatic embryo, matches the hypothetical protein At5g50240, that presents similar features to L-isoaspartyl-O-methyltransferase (PIMT), an enzyme able to repair abnormal isoaspartyl (isoAsp) residues in proteins under normal physiological conditions (Thapar et al, 2002). In plants, L-iso-Asp methyltransferase activity has been identified in monocots, dicots, and green algae (Mudgett and Clarke 1994), although some PIMT enzymatic activity is detectable in a few non-seed tissues (Thapar et al 2002).…”

How Oligomerization Contributes to the Thermostability of an Archaeon Protein

“…The proteins isolated from these organisms are usually more thermostable than homologous proteins from mesophiles, despite the fact that they have similar three-dimensional structures and identical catalytic mechanisms to the mesophilic proteins as well as sequence homologies of 40 -85% (1). Extensive studies have focused on identifying key determinants or common factors responsible for the thermostability of thermophile proteins, and the following structural features have been proposed as responsible for the high thermostability: decreased solvent-exposed surface area (2), increased polar interactions at the molecular surface (3-10), higher packing density within the protein (10 -12), greater core hydrophobicity (10, 13-15), shorter surface loops (11, 16), and more hydrogen bonds (7,10,(17)(18)(19) relative to those of ordinary proteins as well as oligomerization (10, 20 -25).The protein L-isoaspartyl-O-methyltransferase (PIMT 1 ; EC 2.1.1.77) functions as an enzyme in the repair of age-damaged proteins in which asparagines and aspartates have been spontaneously deamidized and isomerized into L-isoaspartyl residues; it catalyzes S-adenosylmethionine (AdoMet)-dependent methylation of the ␣-carboxyl group of the L-isoaspartyl residues to form L-iso-Asp-␣-methyl ester (26,27). 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 protein L-isoaspartyl-O-methyltransferase (PIMT 1 ; EC 2.1.1.77) functions as an enzyme in the repair of age-damaged proteins in which asparagines and aspartates have been spontaneously deamidized and isomerized into L-isoaspartyl residues; it catalyzes S-adenosylmethionine (AdoMet)-dependent methylation of the ␣-carboxyl group of the L-isoaspartyl residues to form L-iso-Asp-␣-methyl ester (26,27). 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.…”