Identification and characterization of a novel class of hydrogen peroxide targets in escherichia coli

Sobota, Jason

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Description

Title

Identification and characterization of a novel class of hydrogen peroxide targets in escherichia coli

Author(s)

Sobota, Jason

Issue Date

2014-05-30T17:04:07Z

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.
• Kehl-Fie, Thomas
• Slauch, James M.

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• hydrogen peroxide
• mononuclear iron enzymes
• oxidative stress
• ribulose 5-phosphate 3-epimerase (Rpe)
• 3-Deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase

Abstract

In nature, microorganisms are constantly exposed to micromolar levels of hydrogen peroxide (H2O2) from sources such as host defenses or byproducts of chemical processes. This constant threat raises two major questions: what biomolecules are damaged by H2O2, and how do cells defend themselves against it? Using Escherichia coli strains that are devoid of peroxide scavengers (Hpx-), I was able to study the effects of protracted, low-grade H2O2 stress. In this study, a novel class of H2O2-sensitive enzymes is described. I discovered that Hpx- cells were unable to metabolize gluconate via the pentose phosphate pathway. By assaying activity of various enzymes in the pathway, one protein, ribulose 5-phosphate 3-epimerase (Rpe), which requires a solvent-exposed divalent metal for activity, was found to be sensitive to H2O2 both in vivo and in vitro. Although Zn, Mn, Fe, and Co were found to activate Rpe to varying degrees, only Rpe loaded with Fe was sensitive to H2O2, which suggests that Fe is the metal that activates Rpe under normal conditions. In addition, Mn supplements were found to protect Rpe from damage by H2O2 in vivo, probably by replacing Fe in the active site. Growth studies also revealed that Hpx- cells are auxotrophic for aromatic amino acids. Less than 0.3 μM H2O2 is necessary to cause this phenotype, making it the most sensitive pathway known. This auxotrophy can be suppressed by adding shikimic acid, an intermediate in the common pathway for aromatic amino acid synthesis (the shikimic acid pathway), as well as Mn2+. I have found that the first enzyme in the shikimic acid pathway (DAHP synthase), which has been shown to have the ability to use Fe2+ as a cofactor, loses activity in Hpx- cells grown in the presence of oxygen. This failure is due to accumulation of damage to DAHP synthase by a Fenton reaction between H2O2 and the active-site Fe atom. This damage results in pathway failure and an inability to synthesize aromatic compounds. The cell attempts to compensate by inducing DAHP synthase transcription, but this is not enough. Mn2+ can replace the Fe in the protein, allowing growth, but still cannot fully activate DAHP synthase to wild-type levels. Thus, Fenton chemistry remains the sole mode of H2O2 toxicity currently known, but the scope of targets of the Fenton reaction is increasing, as many proteins, such as Rpe and DAHP synthase, may use Fe as a mononuclear metal cofactor. Certain aspects of protection against damage by H2O2 are becoming clearer. While the OxyR response increases production of H2O2 scavenging enzymes, it also plays a role in metal homeostasis through regulation of MntH and Dps. These proteins work to increase Mn concentrations, while sequestering Fe in the stressed cell, respectively. This process allows Mn to metallate and thereby protect enzymes which would otherwise be vulnerable to damage by H2O2. Therefore, adjusting metal availability is an important defense mechanism against H2O2, given that enzymes that use divalent metals are significant targets. Each mononuclear Fe-containing enzyme studied thus far could be protected from oxidation by addition of Mn2+, which may help to explain why cells induce Mn import during H2O2 stress.

Graduation Semester

2014-05

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Copyright 2014 Jason Michael Sobota

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