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Please use this identifier to cite or link to this item: http://repository.iitr.ac.in/handle/123456789/1322
Title: In vitro metal catalyzed oxidative stress in DAH7PS: Methionine modification leads to structure destabilization and induce amorphous aggregation
Authors: Sharma A.
Kumar V.
Chatrath A.
Dev A.
Prasad, Ramasare A.
Sharma, Ashwani Kumar
Tomar, Shailly
Kumar, Pravindra R.Manish
Published in: International Journal of Biological Macromolecules
Abstract: The first committed step of the shikimate pathway is catalyzed by a metalloenzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAH7PS), which exhibits vulnerability to the oxidative stress. DAH7PS undergoes inactivation in multiple ways in the presence of redox metal, H2O2, and superoxide. The molecular mechanism and susceptibility of its inactivation might differ in different organisms and are presently unclear. In the present work, we have cloned, expressed and purified a DAH7PS from Providencia alcalifaciens (PaDAH7PS). The oligomeric state and effect of redox metal treatment on its stability were analyzed through the size exclusion chromatography. The FTIR, MALDI-TOF/TOF-MS studies revealed that methionine residues were modified to methionine sulfoxide in PaDAH7PS. During oxidation, PaDAH7PS is altered into partially folded protein and unfolded states as determined by CD and Fluorescence studies. A significant loss in enzymatic activity of PaDAH7PS was determined and the formation of amorphous aggregates was visualized using AFM imaging and also confirmed by ThT binding based assay. This is the first report where we have shown a hexameric DAH7PS and the methionine residues of PaDAH7PS get oxidize in the presence of oxidative stress. The partially folded and unfolded oligomeric states with high ?-content of PaDAH7PS might be the critical precursors for aggregation. © 2017 Elsevier B.V.
Citation: International Journal of Biological Macromolecules(2018), 106(): 1089-1106
URI: https://doi.org/10.1016/j.ijbiomac.2017.08.105
http://repository.iitr.ac.in/handle/123456789/1322
Issue Date: 2018
Publisher: Elsevier B.V.
Keywords: 3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAH7PS)
Methionine modification
Partially folded protein
Protein aggregation
Providencia alcalifaciens
Shikimate pathway
ISSN: 1418130
Author Scopus IDs: 54795869500
57203774869
57195521729
55620085000
16690394400
57214355701
57203506001
55064809000
Author Affiliations: Sharma, A., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Kumar, V., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Chatrath, A., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Dev, A., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Prasad, R., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Sharma, A.K., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Tomar, S., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Kumar, P., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Funding Details: AS would like to thank MHRD; VK and AC would like to thank CSIR; AD would like to thank DBT for providing research fellowship. The author would like to thank Macromolecular Crystallographic Unit, NMR facility, at Institute Instrumentation Centre (IIC), IIT Roorkee for providing Size exclusion chromatography and Fluorescence study facilities, respectively. Author also would like to thank Department of Chemistry, IIT Roorkee for providing FTIR facility.
Corresponding Author: Kumar, P.; Department of Biotechnology, Indian Institute of Technology RoorkeeIndia; email: pravindrak.iitr@gmail.com
Appears in Collections:Journal Publications [BT]

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