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.

journal_name

Biochimie

journal_title

Biochimie

authors

Chatterjee T,Pal A,Chakravarty D,Dey S,Saha RP,Chakrabarti P

doi

10.1016/j.biochi.2012.12.013

subject

Has Abstract

pub_date

2013-04-01 00:00:00

pages

912-21

issue

4

eissn

0300-9084

issn

1638-6183

pii

S0300-9084(12)00504-4

journal_volume

95

pub_type

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