title: Crystal structure and activity of protein L-isoaspartyl-O-methyltransferase from Vibrio cholerae, and the effect of AdoHcy binding.
abstract: The repair enzyme Protein L-isoaspartyl-O-methyltransferase (PIMT) is widely distributed in various organisms. PIMT catalyzes S-adenosylmethionine (AdoMet) dependent methylation of abnormal L-isoaspartyl residues, formed by the deamidation of asparagines and isomerization of aspartates. We report the crystal structure of PIMT of Vibrio cholerae (VcPIMT), the aetiological agent for cholera, complexed with the demethylated cofactor S-adenosyl-L-homocysteine (AdoHcy) to 2.05 Å resolution. A stretch of residues (39-58), lining the substrate-binding site, is disordered. Urea-induced unfolding free energy for apo and VcPIMT-AdoHcy complex reveals greater stability for the cofactor-bound protein. The kinetic parameters for the methyltransferase activity of the recombinant VcPIMT was determined using a continuous spectrophotometric color-based assay using the peptide substrate [VYP(L-isoD)HA]. The enzyme exhibited activity higher than the Escherichia coli enzyme and closer to those from thermophilic bacteria and the mammalian source. The association constant for substrate binding is 2.29 × 10(6) M(-1), quite similar to that for AdoHcy. The crystal structure and the model of the peptide-bound structure indicate that the majority of the interactions used for cofactor/substrate binding are provided by the main-chain atoms. Evolutionary relationships derived based on a phylogenetic tree constructed using the PIMT sequences are in conformity with the crystal structures of nine AdoHcy-bound PIMTs.
authors.affiliation: Department of Biochemistry, Bose Institute, P1/12, CIT Scheme VIIM, Kolkata 700054, India.
authors.forename: Tanaya
authors.initials: T
authors.lastname: Chatterjee
authors.affiliation: Bioinformatics Centre, Bose Institute, P1/12, CIT Scheme VIIM, Kolkata 700054, India.
authors.forename: Debadrita
authors.initials: D
authors.lastname: Mukherjee
authors.affiliation: Department of Biotechnology, West Bengal University of Technology, Kolkata 700064, India.
authors.forename: Mousumi
authors.initials: M
authors.lastname: Banerjee
authors.affiliation: Department of Physics, Bose Institute, 93/1APC Road, Kolkata 700009, India.
authors.forename: Barun K
authors.initials: BK
authors.lastname: Chatterjee
authors.affiliation: Department of Biochemistry, Bose Institute, P1/12, CIT Scheme VIIM, Kolkata 700054, India; Bioinformatics Centre, Bose Institute, P1/12, CIT Scheme VIIM, Kolkata 700054, India. Electronic address: pinak@jcbose.ac.in.
authors.forename: Pinak
authors.initials: P
authors.lastname: Chakrabarti
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D011485:Protein Binding; D011487:Protein Conformation; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*; D058767:Protein Unfolding; D012435:S-Adenosylhomocysteine / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry; D014734:Vibrio cholerae / Q000201:enzymology*
chemical_list: D014508:Urea; D012435:S-Adenosylhomocysteine; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase
keywords: AdoHcy binding; Crystal structure; Enzyme activity; Methyltransferase; Protein repair