Purification, gene cloning, and sequence analysis of an L-isoaspartyl protein carboxyl methyltransferase from Escherichia coli

Fu, June C.; Ding, Li; Clarke, Steven

doi:10.1016/s0021-9258(18)98723-5

Cited by 66 publications

(14 citation statements)

References 67 publications

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“…Kinetic Constant Determinations of the E. coli L-Isoaspartyl Methyltransferase. Methyltransferase-overproducing E. coli HB101 cells containing extrachromosomal copies of the bacterial L-isoaspartyl methyltransferase on the plasmid pMMkatFl (Fu et al, 1991) were grown overnight in 1 L of Terrific Broth (Sambrook et al, 1989). The cells were pelleted and disrupted with a French press at 16000 psi, and cytosolic extracts were prepared as described (Fu et al, 1991).…”

Section: Methodsmentioning

confidence: 99%

“…Methyltransferase-overproducing E. coli HB101 cells containing extrachromosomal copies of the bacterial L-isoaspartyl methyltransferase on the plasmid pMMkatFl (Fu et al, 1991) were grown overnight in 1 L of Terrific Broth (Sambrook et al, 1989). The cells were pelleted and disrupted with a French press at 16000 psi, and cytosolic extracts were prepared as described (Fu et al, 1991). Methyltransferase assays were carried out for 20 min at 37 °C with 8 pL of cytosol (300 pmol min-1 mL-1; specific activity of 3.4 pmol min-1 mg-1) as described above.…”

Section: Methodsmentioning

confidence: 99%

“…The Km and Vmax values represent mean and the standard error calculated by the program from the non-linear curve fit. * Fu et al (1991) and J. Fu, unpublished data. " Values at 25 °C from Mudgett and Clarke (1993).…”

Section: Gaaaqgacitcagrricxjrcnicaactitactojititliaactacaagaactitggca...mentioning

confidence: 97%

“…Substrate Specificity of C. elegans L-Isoaspartyl Methyltransferase. Although all of the previously characterized L-isoaspartyl methyltransferases recognize a variety of lisoaspartyl containing peptides, their affinity for these substrates are variable [ et al, 1984) methyltransferase, it does not appear that either the E. coli (Fu et al, 1991) or the wheat enzyme (Mudgett & Clarke, 1993) is capable of methylating D-aspartyl residues. We thus decided to compare the ability of the C. elegans recombinant enzyme to recognize both L-isoaspartyl, and D-aspartyl-containing peptides.…”

Section: Gaaaqgacitcagrricxjrcnicaactitactojititliaactacaagaactitggca...mentioning

confidence: 99%

“…L-Isoaspartyl methyltransferase activity has been detected in a broad spectrum of organisms, including Gram-negative bacteria (O'Connor & Clarke, 1985;Fu et al, 1991; Li & Clarke, 1992a), protozoans (R. Kagan, unpublished data), fungi (Johnson et al, 1991;R. Kagan, unpublished data), plants (Johnson et al, 1991; Mudgett & Clarke, 1993), ©…”

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Protein L-Isoaspartyl Methyltransferase from the Nematode Caenorhabditis elegans: Genomic Structure and Substrate Specificity

Kagan¹,

Clarke²

1995

Biochemistry

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Gaaaqgacitcagrricxjrcnicaactitactojititliaactacaagaactitggca...mentioning

confidence: 97%

Section: Gaaaqgacitcagrricxjrcnicaactitactojititliaactacaagaactitggca...mentioning

confidence: 99%

mentioning

confidence: 99%

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Protein L-Isoaspartyl Methyltransferase from the Nematode Caenorhabditis elegans: Genomic Structure and Substrate Specificity

Kagan¹,

Clarke²

1995

Biochemistry

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Comparative X-ray analysis of the un-liganded fosfomycin-target MurA

Eschenburg¹,

Schönbrunn²

2000

Proteins

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MurA, an essential enzyme for the synthesis of the bacterial cell wall, follows an induced-fit mechanism. Upon substrate binding, the active site forms in the interdomain cleft, involving movements of the two domains of the protein and a reorientation of the loop Pro112-Pro121. We compare two structures of un-liganded MurA from Enterobacter cloacae: a new orthorhombic form, solved to 1.80 A resolution, and a monoclinic form, redetermined to 1.55 A resolution. In the monoclinic form, the loop Pro112-Pro121 stretches into solvent, while in the new form it adopts a winded conformation, thereby reducing solvent accessibility of the critical residue Cys115. In the interdomain cleft a network of 27 common water molecules has been identified, which partially shields negative charges in the cleft and stabilizes the orientation of catalytically crucial residues. This could support substrate binding and ease domain movements. Near the hinge region an isoaspartyl residue has been recognized, which is the product of post-translational modification of the genetically encoded Asn67-Gly68. The homogeneous population with L-isoaspartate in both structures suggests that the modification in Enterobacter cloacae MurA is not a mere aging defect but rather the result of a specific in vivo process.

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Targeted Gene Disruption of theCaenorhabditis elegansl-Isoaspartyl Protein Repair Methyltransferase Impairs Survival of Dauer Stage Nematodes

Kagan

Niewmierzycka

Clarke

1997

Archives of Biochemistry and Biophysics

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Purification, gene cloning, and sequence analysis of an L-isoaspartyl protein carboxyl methyltransferase from Escherichia coli

Cited by 66 publications

References 67 publications

Protein L-Isoaspartyl Methyltransferase from the Nematode Caenorhabditis elegans: Genomic Structure and Substrate Specificity

Protein L-Isoaspartyl Methyltransferase from the Nematode Caenorhabditis elegans: Genomic Structure and Substrate Specificity

Comparative X-ray analysis of the un-liganded fosfomycin-target MurA

Targeted Gene Disruption of theCaenorhabditis elegansl-Isoaspartyl Protein Repair Methyltransferase Impairs Survival of Dauer Stage Nematodes

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