Expression, purification, and characterization of the protein repair l-isoaspartyl methyltransferase from Arabidopsis thaliana.
Protein l-isoaspartate (d-aspartate) O-methyltransferase (EC 2.1.1. 77) is a repair enzyme that methylates abnormal l-isoaspartate residues in proteins which arise spontaneously as a result of aging. This enzyme initiates their conversion back into the normal l-aspartate form by a methyl esterification reaction. Previously, partial cDNAs of this enzyme were isolated from the higher plant Arabidopsis thaliana. In this study, we report the cloning and expression of a full-length cDNA of l-isoaspartyl methyltransferase from A. thaliana into Escherichia coli under the P(BAD) promoter, which offers a high level of expression under a tight regulatory control. The enzyme is found largely in the soluble fraction. We purified this recombinant enzyme to homogeneity using a series of steps involving DEAE-cellulose, gel filtration, and hydrophobic interaction chromatographies. The homogeneous enzyme was found to have maximum activity at 45 degrees C and a pH optimum from 7 to 8. The enzyme was found to have a wide range of affinities for l-isoaspartate-containing peptides and displayed relatively poor reactivity toward protein substrates. The best methyl-accepting substrates were KASA-l-isoAsp-LAKY (K(m) = 80 microM) and VYP-l-isoAsp-HA (K(m) = 310 microM). We also expressed the full-length form and a truncated version of this enzyme (lacking the N-terminal 26 amino acid residues) in E. coli under the T7 promoter. Both the full-length and the truncated forms were active, though overexpression of the truncated enzyme led to a complete loss of activity.
Thapar
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Department of Chemistry and Biochemistry, Molecular Biology Institute, Los Angeles, California 90095, USA.
Clarke
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D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D002845:Chromatography; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D013696:Temperature
D010940:Plant Proteins; D011994:Recombinant Proteins; D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase