title: Protein l-isoaspartyl-O-methyltransferase of Vibrio cholerae: interaction with cofactors and effect of osmolytes on unfolding.
abstract: Protein l-isoaspartyl-O-methyltransferase (PIMT) is an ubiquitous enzyme widely distributed in cells and plays a role in the repair of deamidated and isomerized proteins. In this study, we show that this enzyme is present in cytosolic extract of Vibrio cholerae, an enteric pathogenic Gram-negative bacterium and is enzymatically active. Additionally, we focus on the detailed biophysical characterization of the recombinant PIMT from V. cholerae to gain insight into its structure, stability and the cofactor binding. The equilibrium denaturation of PIMT has been studied using tryptophan fluorescence and CD spectroscopy. The far- and near-UV CD, as well as fluorescence experiments reveal the presence of a non-native intermediate in the folding pathway. Binding of the hydrophobic fluorescent probe, bis-ANS, to the intermediate occurs with high affinity because of the exposure of the hydrophobic clusters during the unfolding process. The existence of the probable intermediate has also been confirmed from limited tryptic digestion and DLS experiments. The protein shows higher binding affinity for AdoHcy, in comparison to AdoMet, and the binding increases the midpoint of thermal unfolding by 6 and 5 °C, respectively. Modeling and molecular dynamics simulations also support the higher stability of the protein in presence of AdoHcy.
authors.affiliation: Department of Biochemistry, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700054, India. tanaya_chatterjee@yahoo.com
authors.forename: Tanaya
authors.initials: T
authors.lastname: Chatterjee
authors.forename: Aritrika
authors.initials: A
authors.lastname: Pal
authors.forename: Devlina
authors.initials: D
authors.lastname: Chakravarty
authors.forename: Sucharita
authors.initials: S
authors.lastname: Dey
authors.forename: Rudra P
authors.initials: RP
authors.lastname: Saha
authors.forename: Pinak
authors.initials: P
authors.lastname: Chakrabarti
mesh_terms: D003067:Coenzymes / Q000378:metabolism*; D005990:Glycerol / Q000494:pharmacology; D019791:Guanidine / Q000494:pharmacology; D008744:Methylamines / Q000494:pharmacology; D008958:Models, Molecular; D009995:Osmosis / Q000187:drug effects*; D011487:Protein Conformation; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase / Q000737:chemistry* / Q000378:metabolism*; D058767:Protein Unfolding / Q000187:drug effects*; D012436:S-Adenosylmethionine / Q000378:metabolism; D012997:Solvents / Q000737:chemistry; D013312:Stress, Physiological; D013816:Thermodynamics; D014364:Tryptophan; D014734:Vibrio cholerae / Q000201:enzymology* / Q000502:physiology
chemical_list: D003067:Coenzymes; D008744:Methylamines; D012997:Solvents; D012436:S-Adenosylmethionine; D014364:Tryptophan; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; C005855:trimethyloxamine; D019791:Guanidine; D005990:Glycerol