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 degreesC 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 muM) and VYP-L-isoAsp-HA (K-m = 310 muM). 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.