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Heme A synthase from Bacillus subtilis and Aeropyrum pernix

Lewin, Anna LU (2008)
Abstract
Respiration in animals, plants and many bacteria is dependent on heme A, which functions as a prosthetic group in a-type cytochromes (terminal energy-transducing oxidases in the electron transport chain that reduce molecular oxygen to water). Heme A synthase catalyses a chemically very demanding reaction, the conversion of one specific methyl side-group of heme O to a formyl group of heme A. This thesis addresses the structure, evolution and enzyme reaction mechanism of heme A synthase. In the experimental work, the heme A synthase from the gram-positive, soil-bacterium, Bacillus subtilis and the hyper-thermophilic aerobic archaeon Aeropyrum pernix were exploited as model enzymes. In addition to increased knowledge about heme A synthesis,... (More)
Respiration in animals, plants and many bacteria is dependent on heme A, which functions as a prosthetic group in a-type cytochromes (terminal energy-transducing oxidases in the electron transport chain that reduce molecular oxygen to water). Heme A synthase catalyses a chemically very demanding reaction, the conversion of one specific methyl side-group of heme O to a formyl group of heme A. This thesis addresses the structure, evolution and enzyme reaction mechanism of heme A synthase. In the experimental work, the heme A synthase from the gram-positive, soil-bacterium, Bacillus subtilis and the hyper-thermophilic aerobic archaeon Aeropyrum pernix were exploited as model enzymes. In addition to increased knowledge about heme A synthesis, the research results contribute to our understanding of some mitochondria-linked diseases, bacterial physiology, membrane protein evolution, and enzyme catalysis.



Major results of the thesis: (i) A shortened, fully active, mutant of B. subtilis heme A synthase polypeptide (CtaA), obtained using promoted evolution, was shown to have essentially wild-type properties. (ii) A novel (compact) variant of heme A synthase polypeptide (cCtaA), of about half the size of CtaA, was identified in A. pernix. Some properties of the isolated enzyme, produced in Escherichia coli, were determined. (iii) Using a site-specific mutation approach, roles of invariant histidine and cysteine residues in the heme A synthases were analyzed. Some histidine residues probably function as axial heme ligands and cysteine residues can form disulfide bonds in the enzyme. (iv) A procedure for in vitro synthesis of the A. pernix cCtaA membrane protein in the presence of detergent was established. And (v) A system, LALA, for glycerol- or glycerol-3-phosphate dependent gene expression in gram-positive bacteria was constructed and validated in B. subtilis. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Glerum, Moira, Dept of Medical genetics, University of Alberta, Edmonton
organization
publishing date
type
Thesis
publication status
published
subject
keywords
heme A synthesis, heme A, heme A synthase, CtaA, cCtaA, cytochrome a, Aeropyrum pernix, Bacillus subtilis
defense location
Biology Lecture Hall, Sölvegatan 35
defense date
2008-05-23 09:30:00
ISBN
978-91-85067-39-8
language
English
LU publication?
yes
additional info
The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Department of Cell and Organism Biology (Closed 2011.) (011002100)
id
ccaf1f73-9b33-498f-ae2b-b1831e298555 (old id 1146073)
date added to LUP
2016-04-04 14:35:29
date last changed
2018-11-21 21:21:11
@phdthesis{ccaf1f73-9b33-498f-ae2b-b1831e298555,
  abstract     = {{Respiration in animals, plants and many bacteria is dependent on heme A, which functions as a prosthetic group in a-type cytochromes (terminal energy-transducing oxidases in the electron transport chain that reduce molecular oxygen to water). Heme A synthase catalyses a chemically very demanding reaction, the conversion of one specific methyl side-group of heme O to a formyl group of heme A. This thesis addresses the structure, evolution and enzyme reaction mechanism of heme A synthase. In the experimental work, the heme A synthase from the gram-positive, soil-bacterium, Bacillus subtilis and the hyper-thermophilic aerobic archaeon Aeropyrum pernix were exploited as model enzymes. In addition to increased knowledge about heme A synthesis, the research results contribute to our understanding of some mitochondria-linked diseases, bacterial physiology, membrane protein evolution, and enzyme catalysis.<br/><br>
<br/><br>
Major results of the thesis: (i) A shortened, fully active, mutant of B. subtilis heme A synthase polypeptide (CtaA), obtained using promoted evolution, was shown to have essentially wild-type properties. (ii) A novel (compact) variant of heme A synthase polypeptide (cCtaA), of about half the size of CtaA, was identified in A. pernix. Some properties of the isolated enzyme, produced in Escherichia coli, were determined. (iii) Using a site-specific mutation approach, roles of invariant histidine and cysteine residues in the heme A synthases were analyzed. Some histidine residues probably function as axial heme ligands and cysteine residues can form disulfide bonds in the enzyme. (iv) A procedure for in vitro synthesis of the A. pernix cCtaA membrane protein in the presence of detergent was established. And (v) A system, LALA, for glycerol- or glycerol-3-phosphate dependent gene expression in gram-positive bacteria was constructed and validated in B. subtilis.}},
  author       = {{Lewin, Anna}},
  isbn         = {{978-91-85067-39-8}},
  keywords     = {{heme A synthesis; heme A; heme A synthase; CtaA; cCtaA; cytochrome a; Aeropyrum pernix; Bacillus subtilis}},
  language     = {{eng}},
  school       = {{Lund University}},
  title        = {{Heme A synthase from Bacillus subtilis and Aeropyrum pernix}},
  year         = {{2008}},
}