Heme Protein Biogenesis - Catalase in Enterococcus faecalis

Michael Baureder

Research output: ThesisDoctoral Thesis (compilation)

Abstract

Heme proteins form a large and diverse group of proteins which are involved in a variety of biological functions. The heme prosthetic group enables them to carry out redox reactions, transport electrons, bind gaseous molecules, and function as sensors. Despite their importance only little is generally known about heme protein biogenesis.
The Gram-positive bacterium Enterococcus faecalis is found in the gastrointestinal tract of mammals and is an opportunistic pathogen. E. faecalis cannot synthesize heme and does not require heme for growth. When supplied with heme, this bacterium produces two heme proteins; one catalase and one cytochrome bd. These properties of E. faecalis have been exploited in this work to study the physiological role and biogenesis of catalase.
Catalase was found to contribute to resistance against exogenous and endogenous hydrogen peroxide stress. It is shown that the gene for catalase, katA, is expressed independently of heme in the growth medium. KatA protein was found in cells growing in heme-free medium but was degraded in stationary growth phase unless heme was supplied. These and other findings were used to devise a procedure for the purification of apo-catalase polypeptide. It is demonstrated in vitro with isolated apo-catalase and heme that catalase can be de novo assembled. The obtained catalase contained stoichiometric amounts of heme but did not show full enzyme activity. These and other results suggested that the in vitro assembled catalase is stalled at an intermediate state and that one or more soluble cell factors are needed to complete assembly or activation of the enzyme. To find novel factors important for catalase assembly, two constructed libraries of transposon-insertion mutants were screened for catalase deficient mutants. In this way ten independent katA mutations were isolated but no factors (genes) with a dedicated essential role for catalase biogenesis or heme trafficking were revealed. However, the screen indicated nine genes, distributed over five different chromosomal loci, which apparently indirectly affect expression of catalase in E. faecalis.
Original languageEnglish
QualificationDoctor
Awarding Institution
  • Molecular Cell Biology
Supervisors/Advisors
  • Hederstedt, Lars, Supervisor
Award date2012 Apr 12
Publisher
ISBN (Print)978-91-7473-288-7
Publication statusPublished - 2012

Bibliographical note

Defence details

Date: 2012-04-12
Time: 09:30
Place: Biology Building Lecture Hall, Sölvegatan 35, 223 62 Lund

External reviewer(s)

Name: Hartke, Axel
Title: Dr.
Affiliation: Université de Caen

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Subject classification (UKÄ)

  • Biological Sciences

Free keywords

  • Enterococcus faecalis
  • heme proteins
  • protein assembly
  • oxidative stress
  • catalase
  • cytochrome

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