Protein L-isoaspartyl methyltransferase from the nematode Caenorhabditis elegans: genomic structure and substrate specificity.
We identified a protein L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77) in the nematode worm Caenorhabditis elegans. The methylation of abnormal L-isoaspartyl residues by this enzyme can lead to their conversion to L-aspartyl residues and represents a protein repair step for polypeptides damaged by spontaneous reactions during the aging process. We show that the levels of this enzyme increase 2-fold in C. elegans in the dauer larval form, a developmental stage where the organism can survive for extended periods of time. Utilizing degenerate oligonucleotide primers derived from conserved amino acid sequences of mammalian, plant, and bacterial L-isoaspartyl methyltransferases and PCR amplification, we made DNA probes that allowed us to obtain cDNA and genomic DNA clones encoding this enzyme in the nematode. The deduced amino acid sequence is 53% identical to the human enzyme and 29% identical to the Escherichia coli enzyme. Overexpression of the cDNA for the C. elegans enzyme in E. coli gave an active product with micromolar Km values for L-isoaspartyl-containing peptide substrates and for the methyl donor S-adenosyl-L-methionine. No methylation of D-aspartyl-containing peptides was detected under conditions where the human enzyme catalyzed this reaction, suggesting that the ability to methylate D-aspartyl residues in addition to L-isoaspartyl residues was a later evolutionary adaptation of this enzyme. The C. elegans gene for the methyltransferase, designated pcm-1, was mapped to a single site in a 31 kb region in the central portion of chromosome V. The gene is 3.2 kb in length and includes six introns. Although much smaller, its genomic organization is similar to that of the corresponding mouse gene, with identically positioned intron--exon splice junctions at five of seven sites. We propose that this gene plays an important role in facilitating the long term survival of this organisms.
Kagan
R M
RM
Department of Chemistry and Biochemistry, University of California, Los Angeles 90095-1569, USA.
Clarke
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D017173:Caenorhabditis elegans / Q000201:enzymology*; D002470:Cell Survival / Q000235:genetics; D002874:Chromosome Mapping; D002875:Chromosomes / Q000235:genetics; D003001:Cloning, Molecular; D016384:Consensus Sequence / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D017931:DNA Primers; D015870:Gene Expression; D017238:Genes, Helminth; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012326:RNA Splicing / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity
D017931:DNA Primers; D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase