University of SaskatchewanHARVEST
  • Login
  • Submit Your Research
  • About
    • About HARVEST
    • Guidelines
    • Browse
      • All of HARVEST
      • Communities & Collections
      • By Issue Date
      • Authors
      • Titles
      • Subjects
      • This Collection
      • By Issue Date
      • Authors
      • Titles
      • Subjects
    • My Account
      • Login
      JavaScript is disabled for your browser. Some features of this site may not work without it.
      View Item 
      • HARVEST
      • Electronic Theses and Dissertations
      • Graduate Theses and Dissertations
      • View Item
      • HARVEST
      • Electronic Theses and Dissertations
      • Graduate Theses and Dissertations
      • View Item

      Characterization of the Thioredoxin System from Colwellia psychrerythraea and Thermosipho africanus

      Thumbnail
      View/Open
      SAHTOUT-DISSERTATION-2021.pdf (8.929Mb)
      Date
      2021-02-05
      Author
      Sahtout, Naheda Mohamad Fayez
      ORCID
      0000-0001-7033-1575
      Type
      Thesis
      Degree Level
      Doctoral
      Metadata
      Show full item record
      Abstract
      The thioredoxin system is a ubiquitous oxidoreductase system that consists of the enzyme thioredoxin reductase, the protein thioredoxin and the cofactor nicotinamide adenine dinucleotide phosphate. The system has been comprehensively studied from many organisms, such as Escherichia coli; however, structural and functional analysis of this system from psychrophilic and thermophilic bacteria has not been as extensive. In this study, the thioredoxin system proteins of a psychrophilic bacterium, Colwellia psychrerythraea, and a thermophilic bacterium, Thermosipho africanus, were characterized using biophysical, biochemical and X-ray crystallography techniques. Analysis of the complete genome sequence of each of these thioredoxin systems suggested the presence of a putative thioredoxin reductase, at least three thioredoxin in Colwellia psychrerythraea, and at least two thioredoxin in Thermosipho africanus. In this study, the identified putative thioredoxin system components were cloned, overexpressed, purified and characterized. Our studies have indicated that the thioredoxin system proteins from Thermosipho africanus were more stable, able to withstand both higher temperatures and higher concentrations of guanidine hydrochloride before denaturing, than those from Escherichia coli, which in turn were more stable than those from Colwellia psychrerythraea. Consistent with these results, kinetic assays indicated that the thioredoxin reductase from Thermosipho africanus had a higher optimal temperature than that from Escherichia coli, which in turn had a higher optimal temperature than that from Colwellia psychrerythraea. To understand and identify the differences that may contribute to these results, X-ray crystallography was used to determine the structure of Thermosipho africanus thioredoxin 1 and thioredoxin reductase. The structure of thioredoxin 1 showed the typical thioredoxin fold, although lacked the N-terminal region that was found in Escherichia coli thioredoxin 1. The structure of thioredoxin reductase resembled closely that from Escherichia coli. Our studies have therefore identified a novel thermophilic and a novel psychrophilic thioredoxin system that structurally and functionally behave like the thioredoxin system from Escherichia coli.
      Degree
      Doctor of Philosophy (Ph.D.)
      Department
      Chemistry
      Program
      Chemistry
      Supervisor
      Sanders, David
      Committee
      Palmer, David; Dmitriev , Oleg; Bowles, Richard; Scott, Robert; Uppalapati, Maruti Chandra
      Copyright Date
      January 2021
      URI
      http://hdl.handle.net/10388/13256
      Subject
      Colwellia psychrerythraea
      Thermosipho africanus
      thioredoxin reductase
      thioredoxin
      protein crystallography
      enzyme catalysis
      biophysical techniques
      protein stability
      protein-protein interactions
      Collections
      • Graduate Theses and Dissertations
      University of Saskatchewan

      University Library

      © University of Saskatchewan
      Contact Us | Disclaimer | Privacy