Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.14/131967
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- Title
- Bacteriochlorophyll biosynthetic enzymes: molecular mechanistic studies on magnesium chelatase and s-adenosyl- L-methionine, magnesium protoporphyrin IX O-methyltransferase
- Related
- Australasian Digital Theses Program
- Publisher
- Australia : Macquarie University
- Date
- 2010
- Author/Creator
- Sawicki, Artur
- Description
- Thesis by publication.
- Description
- Thesis (PhD)--Macquarie University, Faculty of Science, Dept. of Chemistry and Biomolecular Sciences, 2010.
- Description
- Bibliography: p. 165-184.
- Description
- Introduction -- Paper I: Recessiveness and dominance in barley mutants deficient in Mg-chelatase subunit D, an AAA protein involved in chlorophyll biosynthesis -- Paper II: Kinetic analyses of the magnesium chelatase provide insights into the mechanism, structure, and formation of the complex -- Paper III: S-Adenosyl-L-methionine: magnesium-protoporphyrin IX O-methyltransferase from Rhodobacter capsulatus: mechanistic insights and stimulation with phospholipids -- Paper IV: BchJ functions like a magnesium-protoporphyrin IX carrier between magnesium chelatase and S-adenosyl-L-methionine: magnesium-protoporphyrin IX O-methyltransferase in Rhodobacter capsulatus -- Discussion -- Conclusions and future work -- References.
- Description
- The majority of reactions in the bacteriochlorophyll biosynthetic pathway were first elucidated in the 1940-50's. It is only in recent times that molecular mechanisms of the intermediate steps have been determined. The work presented in this thesis is concerned with mechanistic studies of two successive steps of the pathway from Rba. capsulatus. The two enzymes involved are magnesium chelatase (consisting of BchI, BchD, and BchH subunits), and S-adenosyl-L-methionine:magnesium protoporphyrin IX O-methyltransferase (BchM). Their respective reaction mechanisms were analysed separately and shown how they operate in a coupled system. Also studied is the interaction between magnesium chelatase and an unclassified protein in bacteriochlorophyll biosynthesis, BchJ. -- Dominant inhibition of magnesium chelatase activity in vitro with BchD mutants revealed this subunit was oligomeric. Kinetic data indicated that the molar ratio of BchI:BchD was 1:1, while there are ~2 BchH subunits that interacted with each BchI-BchD complex. It was proposed that secondary catalysis of magnesium chelatase required ATPase activity of BchI for the structural reorganization of the BchI-BchD complex and BchH subunit into catalytic-ready configurations. -- O-methyltransferase required the phospholipid, phosphatidylglycerol for stability and optimal enzymatic activity. Enzyme kinetics showed the Km of Mg-proto from Rba. capsulatus O-methyltransferase was approximately two orders of magnitude lower than the plant/algal enzyme, but similar to O-methyltransferase from another photosynthetic bacterium, Chlorobaculum tepidum. The reaction mechanism was random sequential which is comparable to previous studies with O-methyltransferase from Synechocystis. -- Interactions between magnesium chelatase and BchM or BchJ were observed with magnesium chelatase assays. BchM or BchJ removed the product of the magnesium chelatase reaction, magnesium protoporphyrin IX from BchH. There was a 1:1 molar ratio of BchM or BchJ with BchH. BchH-BchM was the dominant interaction, so it is suggested that BchJ could play a role as a porphyrin binding protein.
- Description
- Mode of access: World Wide Web.
- Description
- x, 184 p . ill. (some col.)
- Subject Keyword
- Enzymology
- Subject Keyword
- Microbial enzymes
- Subject Keyword
- Magnesium chelatase
- Subject Keyword
- Chlorophyll synthesis
- Subject Keyword
- Microbiological synthesis
- Subject Keyword
- Microbiological chemistry
- Subject Keyword
- Botanical chemistry
- Subject Keyword
- bacteriochlorophyll
- Subject Keyword
- BchJ
- Subject Keyword
- enzyme mechanisms
- Subject Keyword
- magnesium chelatase
- Subject Keyword
- methyltransferase
- Resource Type
- Thesis PhD
- Organisation
- Macquarie University. Dept. of Chemistry and Biomolecular Sciences
- Identifier
- http://hdl.handle.net/1959.14/131967
- Identifier
- 1451958
- Language
- eng
- Rights
- Copyright disclaimer: http://www.copyright.mq.edu.au
- Rights
- Copyright Artur Sawicki 2010.
- Full Text
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