Magnesium Chelatase: Insights into the first Step of Chlorophyll Biosynthesis
(2006)- Abstract
- The enzyme magnesium chelatase inserts magnesium into protoporphyrin IX (Proto) to produce to magnesium protoporphyrin IX, the first unique intermediate of the chlorophyll biosynthetic pathway. Magnesium chelatase is composed of three distinct proteins termed I (molecular weight ~40 kDa), D (~70 kDa) and H (~140 kDa). Defining the individual properties and structure of the magnesium chelatase components and their role in the reaction mechanism is important for a full understanding of the first step in magnesium tetrapyrrole biosynthesis. The three components of magnesium chelatase show significant conservation at the protein sequence level, which extends from bacteriochlorophyll synthesising purple non-sulfur bacteria and green sulfur... (More)
- The enzyme magnesium chelatase inserts magnesium into protoporphyrin IX (Proto) to produce to magnesium protoporphyrin IX, the first unique intermediate of the chlorophyll biosynthetic pathway. Magnesium chelatase is composed of three distinct proteins termed I (molecular weight ~40 kDa), D (~70 kDa) and H (~140 kDa). Defining the individual properties and structure of the magnesium chelatase components and their role in the reaction mechanism is important for a full understanding of the first step in magnesium tetrapyrrole biosynthesis. The three components of magnesium chelatase show significant conservation at the protein sequence level, which extends from bacteriochlorophyll synthesising purple non-sulfur bacteria and green sulfur bacteria to chlorophyll synthesising eukaryotes and cyanobacteria.
In Paper I, eight mutants of the H gene (Xantha-f) from barley were characterised at the molecular level and provide explanations for the yellow phenotypes of germinating mutant seedlings. In this thesis magnesium chelatase from the photosynthetic bacterium Rhodobacter capsulatus has been used as a model system since much of the pioneering work has been conducted on this organism. Magnesium chelatase requires ATP to insert magnesium into Proto. There has been conflicting results as to the ATPase activity of the H subunit. In Paper II it was demonstrated that ATP hydrolysis can be attributed the I subunit and not the H. The unprecedented discovery of an iron-sulfur cluster in the H subunit of R. capsulatus is described in Paper III. The cysteine motif that coordinates this iron-sulfur cluster is only present in five other facultative proteobacteria and absent in all oxygenic or anaerobic species. The function of this cluster is yet to be established. In Paper IV the first insights into the structure of an H subunit is presented. Electron microscopy and single-particle reconstruction was used to solve the structure in the apo and substrate bound conformations at a resolution of 25 Å, and revealed a conformational change upon Proto binding. Limited proteolysis and construction of truncated H polypeptides provided supporting information to propose a cooperative substrate binding model. The binding of porphyrin to the H subunit was further investigated in Paper V using tryptophan fluorescence quenching to detect a high affinity porphyrin binding site in the nanomolar range. Alanine mutagenesis of the H subunit implicated key residues involved in porphyrin binding and catalysis.
The work presented in this thesis has expanded our understanding of the magnesium chelatase, particularly in respect to the H subunit. With three different proteins and three substrates, it is clear that magnesium chelatase is an elaborate molecular machine that is proving to be far more complicated than ever expected. (Less) - Abstract (Swedish)
- Popular Abstract in Swedish
Enzymet magnesium-kelatas katalyserar bildandet av magnesium-protoporfyrin genom insättning av en magnesiumjon i protoporfyrin. Detta är det första unika steget i tillverkningen av klorofyll hos växter, alger och fotosyntetiska bakterier. Magnesium-kelatas består av tre proteiner som kallas I (vikt 40 kDa), D (70 kDa) och H (140 kDa). Det är viktigt att bestämma dessa proteiners egenskaper och struktur för att förtså detta inledande katalytiska steg i klorofyllsyntesen. De tre proteinerna är starkt evolutionärt konserverade när man jämför motsvarande proteiner från så pass skilda organismer som purpur icke-svavel bakterier och gröna svavel bakterier till eukaryota växter och... (More) - Popular Abstract in Swedish
Enzymet magnesium-kelatas katalyserar bildandet av magnesium-protoporfyrin genom insättning av en magnesiumjon i protoporfyrin. Detta är det första unika steget i tillverkningen av klorofyll hos växter, alger och fotosyntetiska bakterier. Magnesium-kelatas består av tre proteiner som kallas I (vikt 40 kDa), D (70 kDa) och H (140 kDa). Det är viktigt att bestämma dessa proteiners egenskaper och struktur för att förtså detta inledande katalytiska steg i klorofyllsyntesen. De tre proteinerna är starkt evolutionärt konserverade när man jämför motsvarande proteiner från så pass skilda organismer som purpur icke-svavel bakterier och gröna svavel bakterier till eukaryota växter och cyanobakterier.
I artikel I karaktäriseras åtta korn-mutanter som är defekta i H-genen (Xantha-f) på DNA-nivå och deras gula fenotyp kunde därmed förklaras. I den här avhandlingen används ofta magnesium-kelatas från bakterien Rhodobacter capsulatus som modellsystem, då mycket av pionjärarbetet kring magnesium-kelatas har gjort med denna organism. Magnesium-kelatas behöver ATP för att kunna sätta in magnesium i protoporfyrin och det har tidigare föreslagits att H-proteinet skulle kunna omsätta detta ATP. I artikel II visar vi att ATP-hydrolysen bara är associerad till I-proteinet och inte H-proteinet. Den oförutsedda upptäckten av ett järn-svavel-kluster i H-proteinet från R. capsulatus beskrivs i artikel III. Det cystein-motiv som koordinerar järn-svavel-klustret hittas bara i fem fakultativt anaeroba proteobakterier och saknas i aeroba och strikt anaeroba arter. Funktionen av järn-svavel-klustret behöver klargöras. I artikel IV presenteras den första strukturella beskrivningen av H-proteinet. Elektronmikroskopi och ?single-particle? rekonstruering användes för att lösa strukturen med och utan bundet protoporfyrin. De strukturella förändringar proteinet genomgår vid substrat-inbindning visas vid 25 Å upplösning. Dessutom ger proteolysbehandling och analyser av förkortade versioner av H-proteinet bevis för kooperativ bindning av porfyrin-substratet. Bindningen av porfyrin till H-proteinet studeras ytterligare i artikel V med hjälp av tryptofan-fluoroscens och ett bindningsställe med hög affinitet beskrivs. Utbyte av specifika aminosyror i H-proteinet genom riktad mutagenes visar vilka aminosyrer som är viktiga för substratbindning och/eller katalys.
De resultat som presenteras i denna avhandling utökar vår förståelse för magnesium-kelataset och då speciellt H-proteinet. Med tre olika protein-komponenter och tre inblandade substrat, så står det klart att magnesium-kelatas är en högt utvecklad molekylär maskin som är vida mer komplicerad än vad som någonsin kunnat förväntas. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/547117
- author
- Sirijovski, Nick LU
- supervisor
-
- Mats Hansson LU
- opponent
-
- Professor Warren, Martin, Department of Bioscience, University of Kent, U.K.
- organization
- publishing date
- 2006
- type
- Thesis
- publication status
- published
- subject
- keywords
- Electron microscopy, Proteobacteria, Mutants, Rhodobacter capsulatus, Tetrapyrrole, Plant biochemistry, Växtbiokemi, Biochemistry, Metabolism, Biokemi, metabolism, Proteins, enzymologi, enzymology, Proteiner, Chlorophyll biosynthesis, AAA+ proteins, Magnesium chelatase, Barley, ATPase
- pages
- 110 pages
- publisher
- KFS AB
- defense location
- Kemicentrum, Sölvegatan 39, Lund Hörsal B
- defense date
- 2006-09-22 10:00:00
- ISBN
- 978-91-628-6925-0
- language
- English
- LU publication?
- yes
- id
- cd1951c8-d956-4b39-acaf-46260a49e7cb (old id 547117)
- date added to LUP
- 2016-04-04 10:21:08
- date last changed
- 2018-11-21 20:58:16
@phdthesis{cd1951c8-d956-4b39-acaf-46260a49e7cb,
abstract = {{The enzyme magnesium chelatase inserts magnesium into protoporphyrin IX (Proto) to produce to magnesium protoporphyrin IX, the first unique intermediate of the chlorophyll biosynthetic pathway. Magnesium chelatase is composed of three distinct proteins termed I (molecular weight ~40 kDa), D (~70 kDa) and H (~140 kDa). Defining the individual properties and structure of the magnesium chelatase components and their role in the reaction mechanism is important for a full understanding of the first step in magnesium tetrapyrrole biosynthesis. The three components of magnesium chelatase show significant conservation at the protein sequence level, which extends from bacteriochlorophyll synthesising purple non-sulfur bacteria and green sulfur bacteria to chlorophyll synthesising eukaryotes and cyanobacteria.<br/><br>
<br/><br>
In Paper I, eight mutants of the H gene (Xantha-f) from barley were characterised at the molecular level and provide explanations for the yellow phenotypes of germinating mutant seedlings. In this thesis magnesium chelatase from the photosynthetic bacterium Rhodobacter capsulatus has been used as a model system since much of the pioneering work has been conducted on this organism. Magnesium chelatase requires ATP to insert magnesium into Proto. There has been conflicting results as to the ATPase activity of the H subunit. In Paper II it was demonstrated that ATP hydrolysis can be attributed the I subunit and not the H. The unprecedented discovery of an iron-sulfur cluster in the H subunit of R. capsulatus is described in Paper III. The cysteine motif that coordinates this iron-sulfur cluster is only present in five other facultative proteobacteria and absent in all oxygenic or anaerobic species. The function of this cluster is yet to be established. In Paper IV the first insights into the structure of an H subunit is presented. Electron microscopy and single-particle reconstruction was used to solve the structure in the apo and substrate bound conformations at a resolution of 25 Å, and revealed a conformational change upon Proto binding. Limited proteolysis and construction of truncated H polypeptides provided supporting information to propose a cooperative substrate binding model. The binding of porphyrin to the H subunit was further investigated in Paper V using tryptophan fluorescence quenching to detect a high affinity porphyrin binding site in the nanomolar range. Alanine mutagenesis of the H subunit implicated key residues involved in porphyrin binding and catalysis.<br/><br>
<br/><br>
The work presented in this thesis has expanded our understanding of the magnesium chelatase, particularly in respect to the H subunit. With three different proteins and three substrates, it is clear that magnesium chelatase is an elaborate molecular machine that is proving to be far more complicated than ever expected.}},
author = {{Sirijovski, Nick}},
isbn = {{978-91-628-6925-0}},
keywords = {{Electron microscopy; Proteobacteria; Mutants; Rhodobacter capsulatus; Tetrapyrrole; Plant biochemistry; Växtbiokemi; Biochemistry; Metabolism; Biokemi; metabolism; Proteins; enzymologi; enzymology; Proteiner; Chlorophyll biosynthesis; AAA+ proteins; Magnesium chelatase; Barley; ATPase}},
language = {{eng}},
publisher = {{KFS AB}},
school = {{Lund University}},
title = {{Magnesium Chelatase: Insights into the first Step of Chlorophyll Biosynthesis}},
year = {{2006}},
}