Expression, Purification, and Characterization of the Protein Repair l-Isoaspartyl Methyltransferase from Arabidopsis thaliana

Thapar, Nitika; Clarke, Steven

doi:10.1006/prep.2000.1311

Cited by 24 publications

(28 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…We found that the P. furiosus enzyme recognizes these substrates with affinities much higher than those of other prokaryotic organisms and in fact even higher in some cases than those of the human enzyme, previously determined to be the most effective catalyst of these reactions (24). The K m values for the L-isoaspartate peptides were comparable for the P. furiosus and human enzymes, whereas the P. furiosus enzyme recognized the protein ovalbumin with about 8-fold higher affinity than the human enzyme (Table I).…”

Section: Figmentioning

confidence: 68%

“…Additionally, no activity on D-aspartyl residues in short peptides was found with L-isoaspartyl methyltransferases isolated from E. coli (28), T. maritima (10), Arabidopsis (24), and nematodes (13), suggesting that the ability to methylate D-aspartyl residues might be a special adaptation of the methyltransferases in complex and long-lived mammalian species. Our finding here that the P. furiosus enzyme recognizes D-aspartyl residues in peptides with a 120-fold higher affinity than the human enzyme suggests, however, that the ability of cells to recognize spontaneously damaged proteins containing a Substrates were used at a final concentration of 1 mM.…”

Section: Figmentioning

confidence: 99%

“…We have been particularly interested in the enzymes that exist in thermophilic organisms where the rate of spontaneous isomerization and racemization would be expected to be greatly enhanced. Characterization of the enzyme from the eubacterium Thermotoga maritima demonstrated superior recognition of methylaccepting substrates compared with the E. coli, plant, and worm enzymes, but the recognition is still 5-14-fold poorer than that of the human enzyme (10,24). Additionally, the T. maritima enzyme has not been observed to methylate D-aspartyl-containing peptides recognized by the human enzyme (10).…”

mentioning

confidence: 97%

“…For example, the human enzyme recognizes substrates with 40 -1000 times the affinity of the E. coli, nematode, and plant enzymes (24). The functional significance of these differences is not clear, although mathematical simulations of the repair reaction clearly show that repair efficiency is directly related to the affinity of the enzyme for the methyl-acceptor (25).…”

mentioning

confidence: 99%

“…The K m for corresponding synthetic peptides containing D-aspartyl residues in place of L-isoaspartyl residues can be 700 -10,000-fold higher, however. Additionally, no methylation of D-aspartyl peptides has been detected for the E. coli (28), worm (13), and higher plant (24) enzymes. In mammalian cells, it is possible that the methylation of D-aspartyl residues can lead to eventual L-aspartyl formation in a repair reaction similar to that observed for L-isoaspartyl residues, although considerable amounts of D-isoaspartyl residues would also be expected to accumulate (27).…”

mentioning

confidence: 99%

See 4 more Smart Citations

Protein Repair Methyltransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Thapar

Griffith

Yeates

et al. 2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Section: Figmentioning

confidence: 68%

Section: Figmentioning

confidence: 99%

mentioning

confidence: 97%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Protein Repair Methyltransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Thapar

Griffith

Yeates

et al. 2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

Development of Chemical Tools to Monitor and Control Isoaspartyl Peptide Methyltransferase Activity

et al. 2016

View full text Add to dashboard Cite

Abstract:We have established a coupled assay system targeting protein l-isoaspartyl methyltransferase (PIMT), a key enzyme in the metabolism of isoaspartyl peptides and proteins. The system utilizes a fluorogenic peptide probe containing an isoaspartyl residue at the P1' position of the caspase-3 recognition sequence. Following PIMT-catalyzed methyl transfer reaction, the methylated probe is specifically cleaved by caspase-3 to give fluorescence activation. High-throughput screening of our chemical library with this assay system identified PIMT inhibitors that may be useful as leads in the design of chemical probes for controlling PIMT activity.

show abstract

Development of Chemical Tools to Monitor and Control Isoaspartyl Peptide Methyltransferase Activity

et al. 2016

View full text Add to dashboard Cite

We have established a coupled assay system targeting protein l-isoaspartyl methyltransferase (PIMT), a key enzyme in the metabolism of isoaspartyl peptides and proteins. The system utilizes a fluorogenic peptide probe containing an isoaspartyl residue at the P1' position of the caspase-3 recognition sequence. Following PIMT-catalyzed methyl transfer reaction, the methylated probe is specifically cleaved by caspase-3 to give fluorescence activation. High-throughput screening of our chemical library with this assay system identified PIMT inhibitors that may be useful as leads in the design of chemical probes for controlling PIMT activity.

show abstract

Expression, Purification, and Characterization of the Protein Repair l-Isoaspartyl Methyltransferase from Arabidopsis thaliana

Cited by 24 publications

References 48 publications

Protein Repair Methyltransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Protein Repair Methyltransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Development of Chemical Tools to Monitor and Control Isoaspartyl Peptide Methyltransferase Activity

Development of Chemical Tools to Monitor and Control Isoaspartyl Peptide Methyltransferase Activity

Contact Info

Product

Resources

About