Superoxide stress in Escherichia coli

Gu, Mianzhi

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https://hdl.handle.net/2142/44253 Copy

Description

Title

Superoxide stress in Escherichia coli

Author(s)

Gu, Mianzhi

Issue Date

2013-05-24T22:05:35Z

Director of Research (if dissertation) or Advisor (if thesis)

Imlay, James A.

Doctoral Committee Chair(s)

Imlay, James A.

Committee Member(s)

• Cronan, John E.
• Metcalf, William W.
• Kuzminov, Andrei

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• oxidative stress
• superoxide
• redox-cycling drugs
• metalloenzymes
• SoxRS response.

Abstract

Since its discovery in 1969, superoxide (O2-) has been recognized as a primary source of oxidative stress. However, in the last 40 years, the only cellular target of O2- that has been identified is a subfamily of [4Fe-4S] dehydratases. The inactivation of those dehydratases can only explain part of its toxic effects, which implies the existence of other unknown cellular targets. We have recently identified a new class of cellular targets of O2-: non-redox mononuclear iron enzymes. These enzymes employ a ferrous iron atom [Fe(II)] as a solvent-exposed cofactor. Four such enzymes -- threonine dehydrogenase (Tdh), ribulose-5-phosphate 3-epimerase (Rpe), peptide deformylase (PDF), and DAHP synthase -- were shown to be inactivated in the Escherichia coli strain which lacks the ability to scavenge O2- (denoted as SOD- mutant). Moreover, the inactivation of DAHP synthase, the first enzyme in the aromatic amino acid biosynthetic pathway, could partially explain the aromatic amino acid auxotrophy of SOD- mutants. All four enzymes were purified and were demonstrated to be rapidly inactivated by O2- in vitro. The inactivation rate constants were comparable to those with which O2- reacts with [4Fe-4S] dehydratases. It was initially a puzzle how O2- could damage those iron enzymes in vivo, since unlike H2O2 which can form a ferryl radical with the catalytic Fe(II) and damage the polypeptide, O2- is not able to do so. In fact, we found that Tdh and Rpe isolated from the O2--stressed stain were metallated with Zn(II) rather than with Fe(II). Therefore, we propose that O2- oxidizes Fe(II), the oxidized Fe(III) dissociates from the enzyme, and Zn(II) gets the chance to bind. Since Zn(II) binds to these enzymes much more tightly than does iron, it traps the enzymes in the mismetallated form which are much less efficient in catalysis. O2--sensitive [4Fe-4S] dehydratases and mononuclear iron enzymes are present throughout metabolism. Therefore, a number of bacteria and plants exploit this vulnerability: they excrete antimicrobial compounds known as redox-cycling drugs to elevate the production of O2- in their competitors. When E. coli is exposed to these compounds, its SoxR transcription factor is activated by oxidation of its [2Fe-2S] cluster. O2- was initially thought to be the activator of SoxR, because the O2--scavenging enzyme – superoxide dismutase (SOD) is a member of the SoxRS regulon. However, we found that abundant O2- did not effectively activate SoxR in an SOD- mutant, that overproduced SOD could not suppress activation by redox-cycling drugs, and that redox-cycling drugs were able to activate SoxR in anaerobic cells as long as alternative respiratory acceptors were provided. Thus O2- is not the signal that SoxR senses. Indeed, redox-cycling drugs directly oxidized the cluster of purified SoxR in vitro, while O2- did not. Redox-cycling drugs also caused cellular toxicity independent of the production of O2-, as they poisoned E. coli under anaerobic conditions, in part by oxidizing [4Fe-4S] dehydratases. SoxRS affects the expression of nearly 100 genes, most of which do little to ameliorate O2- toxicity. Instead, they focus upon reducing the intracellular levels of redox-cycling drugs. Thus it is physiologically appropriate that the SoxR protein directly senses redox-cycling drugs rather than O2-.

Graduation Semester

2013-05

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http://hdl.handle.net/2142/44253

Copyright and License Information

Copyright 2013 Mianzhi Gu

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