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• W2027401865 abstract "We have identified a gene (iadA) in Escherichia coli encoding a 41-kDa polypeptide that catalyzes the hydrolytic cleavage of L-isoaspartyl, or L-β-aspartyl, dipeptides. We demonstrate at least a 3000-fold purification of the enzyme to homogeneity from crude cytosol. From the amino-terminal amino acid sequence obtained from this preparation, we designed an oligonucleotide that allowed us to map the gene to the 98-min region of the chromosome and to clone and obtain the DNA sequence of the gene. Examination of the deduced amino acid sequence revealed no similarities to other peptidases or proteases, while a marked similarity was found with several dihydroorotases and imidases, reflecting the similarity in the structures of the substrates for these enzymes. Using an E. coli strain containing a plasmid overexpressing this gene, we were able to purify sufficient amounts of the dipeptidase to characterize its substrate specificity. We also examined the phenotype of two E. coli strains where this isoaspartyl dipeptidase gene was deleted. We inserted a chloramphenicol cassette into the disrupted coding region of iadA in both a parent strain (MC1000) and a derivative strain (CL1010) lacking pcm, the gene encoding the L-isoaspartyl methyltransferase involved in the repair of isomerized proteins. We found that the iadA deletion does not result in reduced stationary phase or heat shock survival. Analysis of isoaspartyl dipeptidase activity in the deletion strain revealed a second activity of lower native molecular weight that accounts for approximately 31% of the total activity in the parent strain MC1000. The presence of this second activity may account for the absence of an observable phenotype in the iadA mutant cells. We have identified a gene (iadA) in Escherichia coli encoding a 41-kDa polypeptide that catalyzes the hydrolytic cleavage of L-isoaspartyl, or L-β-aspartyl, dipeptides. We demonstrate at least a 3000-fold purification of the enzyme to homogeneity from crude cytosol. From the amino-terminal amino acid sequence obtained from this preparation, we designed an oligonucleotide that allowed us to map the gene to the 98-min region of the chromosome and to clone and obtain the DNA sequence of the gene. Examination of the deduced amino acid sequence revealed no similarities to other peptidases or proteases, while a marked similarity was found with several dihydroorotases and imidases, reflecting the similarity in the structures of the substrates for these enzymes. Using an E. coli strain containing a plasmid overexpressing this gene, we were able to purify sufficient amounts of the dipeptidase to characterize its substrate specificity. We also examined the phenotype of two E. coli strains where this isoaspartyl dipeptidase gene was deleted. We inserted a chloramphenicol cassette into the disrupted coding region of iadA in both a parent strain (MC1000) and a derivative strain (CL1010) lacking pcm, the gene encoding the L-isoaspartyl methyltransferase involved in the repair of isomerized proteins. We found that the iadA deletion does not result in reduced stationary phase or heat shock survival. Analysis of isoaspartyl dipeptidase activity in the deletion strain revealed a second activity of lower native molecular weight that accounts for approximately 31% of the total activity in the parent strain MC1000. The presence of this second activity may account for the absence of an observable phenotype in the iadA mutant cells." @default.
• W2027401865 created "2016-06-24" @default.
• W2027401865 creator A5082847533 @default.
• W2027401865 creator A5090650225 @default.
• W2027401865 date "1995-02-01" @default.
• W2027401865 modified "2023-09-30" @default.
• W2027401865 title "Purification and Characterization of an Isoaspartyl Dipeptidase from Escherichia coli" @default.
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• W2027401865 doi "https://doi.org/10.1074/jbc.270.8.4076" @default.
• W2027401865 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7876157" @default.
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