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Heme Protein Biogenesis - Catalase in Enterococcus faecalis

Baureder, Michael LU (2012)
Abstract (Swedish)
Popular Abstract in English

Heme is a small red-colored molecule consisting of an iron atom bound to a chemical structure called porphyrin. Heme is essential for many organisms, including humans, because it is a cofactor in a variety of proteins that are needed to sustain metabolism. The probably most famous heme protein is hemoglobin which gives blood its red color. The heme cofactor in hemoglobin binds oxygen to be distributed in the body.

Besides the binding of oxygen heme can confer a protein with one of several other functions. Those are certain chemical reactions needed for the degradation of toxic compounds, the transport of electrons needed for generation of energy from nutrients, and the sensing of oxygen... (More)
Popular Abstract in English

Heme is a small red-colored molecule consisting of an iron atom bound to a chemical structure called porphyrin. Heme is essential for many organisms, including humans, because it is a cofactor in a variety of proteins that are needed to sustain metabolism. The probably most famous heme protein is hemoglobin which gives blood its red color. The heme cofactor in hemoglobin binds oxygen to be distributed in the body.

Besides the binding of oxygen heme can confer a protein with one of several other functions. Those are certain chemical reactions needed for the degradation of toxic compounds, the transport of electrons needed for generation of energy from nutrients, and the sensing of oxygen enabling organisms to adapt their metabolism.

Despite the importance of heme proteins in nature we still do not understand how they are produced in cells, i.e. by what mechanisms the heme group becomes inserted into proteins. One well-studied heme protein is catalase. This protein is responsible for the degradation of toxic hydrogen peroxide in many organisms. Catalase enzymes of many diverse organisms, e.g. bacteria and mammals, are very similar. In this work we have used the bacterium Enterococcus faecalis as a simple model organism to study how catalase is produced and how important this enzyme is for survival of the bacterium.

Catalase is made up of four identical protein subunits that are combined to a functional enzyme in a complex fashion. One heme molecule is placed into each of the four subunits. The research work of this thesis comprises cellular processes needed for formation of functional catalase enzyme.

E. faecalis is resistant to high concentrations of hydrogen peroxide. The reason for that is that this bacterium in addtition to catalase produces several enzymes that degrade hydrogen peroxide. The contribution of catalase to protection against hydrogen peroxide was studied.

The findings presented in this thesis increase our knowledge about the cellular processes needed for production of a heme protein. They also contribute to a better understanding of lactic acid bacteria including E. faecalis which is used within the food industry and which is an opportunistic pathogen that under certain circumstances can cause severe infections. (Less)
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... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Dr. Hartke, Axel, Université de Caen
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Enterococcus faecalis, heme proteins, protein assembly, oxidative stress, catalase, cytochrome
pages
122 pages
publisher
Department of Biology, Lund University
defense location
Biology Building Lecture Hall, Sölvegatan 35, 223 62 Lund
defense date
2012-04-12 09:30
ISBN
978-91-7473-288-7
language
English
LU publication?
yes
id
7cc9ccdc-4dc9-4ede-ac68-e48cd7484b22 (old id 2372161)
date added to LUP
2012-03-16 11:14:54
date last changed
2016-09-19 08:45:13
@misc{7cc9ccdc-4dc9-4ede-ac68-e48cd7484b22,
  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.<br/><br>
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.<br/><br>
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.},
  author       = {Baureder, Michael},
  isbn         = {978-91-7473-288-7},
  keyword      = {Enterococcus faecalis,heme proteins,protein assembly,oxidative stress,catalase,cytochrome},
  language     = {eng},
  pages        = {122},
  publisher    = {ARRAY(0x9865178)},
  title        = {Heme Protein Biogenesis - Catalase in Enterococcus faecalis},
  year         = {2012},
}