Characterization of Rhodobacter capsulatus and barley (Hordeum vulgare L.) magnesium chelatases
(2019) MOBN03 20182Degree Projects in Molecular Biology
- Abstract
- Magnesium chelatase is a heterotrimeric enzyme complex, which catalyzes the insertion of Mg2+ into protoporphyrin IX. The enzyme is composed of three protein subunits BchI, BchD and BchH of approximately 40, 70 and 140 kDa, respectively. In barley (Hordeum vulgare L.) the subunits are referred to as Xantha-H, Xantha-G and Xantha-F. In this study, genes encoding the magnesium chelatase subunits of the purple non-sulfur bacterium Rhodobacter capsulatus and H. vulgare were successfully expressed individually in Escherichia coli. The recombinant proteins were purified and used in enzyme assays. Further, the stability of the proteins at different temperatures and in different buffer solutions with various pH conditions was also tested. Protein... (More)
- Magnesium chelatase is a heterotrimeric enzyme complex, which catalyzes the insertion of Mg2+ into protoporphyrin IX. The enzyme is composed of three protein subunits BchI, BchD and BchH of approximately 40, 70 and 140 kDa, respectively. In barley (Hordeum vulgare L.) the subunits are referred to as Xantha-H, Xantha-G and Xantha-F. In this study, genes encoding the magnesium chelatase subunits of the purple non-sulfur bacterium Rhodobacter capsulatus and H. vulgare were successfully expressed individually in Escherichia coli. The recombinant proteins were purified and used in enzyme assays. Further, the stability of the proteins at different temperatures and in different buffer solutions with various pH conditions was also tested. Protein crystals of R. capsulatus BchI were successfully formed in hanging drops by using 16% polyethylene glycol solution. Magnesium chelatase assays were performed at multiple timelines, temperature gradients and buffers at different pH. Furthermore, magnesium chelatase activity was tested with different combinations of recombinant R. capsulatus and H. vulgare protein subunits in combination with barley chloroplast extract. It was observed that recombinant R. capsulatus protein subunits did not influence the formation of Mg-deuteroporphyrin IX when combined with chloroplast extract whereas addition of recombinant H. vulgare protein subunits stimulated activity. It was also found that recombinant H. vulgare protein subunits showed magnesium chelatase activity by themselves. However, increasing amounts of added recombinant Xantha-G protein subunit decreased the activity. (Less)
- Popular Abstract
- Chlorophyll is one of the most abundant pigment in plants, algae and cyanobacteria. It is synthesized through a multistep pathway (approximately 20 steps) in which protoporphyrin IX (Proto) is the branch-point precursor for both haem and chlorophyll. Mg-chelatase (EC 6.6.1.1) catalyses the insertion of Mg into protoporphyrin IX (Proto) which is the first committed reaction in chlorophyll biosynthesis through the ATP-dependent insertion of Mg2+ into the protoporphyrin IX. In figure 1 it is showed how magnesium is incorporated into the tetrapyrrole ring with the help of ATP. Mg-chelatase is a heterotrimeric enzyme and consists of three subunits; I (~40kDa), D (~70kDa) and H (~140kDa), respectively, and requires ATP for magnesium chelation.... (More)
- Chlorophyll is one of the most abundant pigment in plants, algae and cyanobacteria. It is synthesized through a multistep pathway (approximately 20 steps) in which protoporphyrin IX (Proto) is the branch-point precursor for both haem and chlorophyll. Mg-chelatase (EC 6.6.1.1) catalyses the insertion of Mg into protoporphyrin IX (Proto) which is the first committed reaction in chlorophyll biosynthesis through the ATP-dependent insertion of Mg2+ into the protoporphyrin IX. In figure 1 it is showed how magnesium is incorporated into the tetrapyrrole ring with the help of ATP. Mg-chelatase is a heterotrimeric enzyme and consists of three subunits; I (~40kDa), D (~70kDa) and H (~140kDa), respectively, and requires ATP for magnesium chelation. In plants, the Mg-chelatase genes are recognized as chlI, chlD and chlH. The corresponding proteins are named as ChlI, ChlD and ChlH, respectively. On the other hand, in bacteria that synthesize bacteriochlorophyll, the genes are termed as bchI, bchD and bchH and the proteins are known as BchI, BchD and BchH.
The work in this master’s thesis was mainly focused on the characterization of magnesium chelatase subunits in chlorophyll biosynthesis pathways in barley (Hordeum vulgare) and the bacterium Rhodobacter capsulatus, which is a purple bacterium that can obtain energy through photosynthesis. Starting with the construction of expression plasmids containing the gene of interest the recombinant proteins in Escherichia coli was produced. The stability of each protein in different environments was investigated to observe which conditions are favorable for the proteins. Finally, the interaction between the subunits was observed through enzymatic measurements.
Master’s Degree Project – Molecular Biology 60 credits 2019
Department of Biology, Lund University
Advisor: Professor Mats Hansson
Head of Unit Molecular Cell Biology, Lund University (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/8995233
- author
- Mahdi, Rabab
- supervisor
-
- Mats Hansson LU
- organization
- course
- MOBN03 20182
- year
- 2019
- type
- H2 - Master's Degree (Two Years)
- subject
- language
- English
- id
- 8995233
- date added to LUP
- 2019-09-17 11:22:36
- date last changed
- 2019-09-18 13:30:37
@misc{8995233,
abstract = {{Magnesium chelatase is a heterotrimeric enzyme complex, which catalyzes the insertion of Mg2+ into protoporphyrin IX. The enzyme is composed of three protein subunits BchI, BchD and BchH of approximately 40, 70 and 140 kDa, respectively. In barley (Hordeum vulgare L.) the subunits are referred to as Xantha-H, Xantha-G and Xantha-F. In this study, genes encoding the magnesium chelatase subunits of the purple non-sulfur bacterium Rhodobacter capsulatus and H. vulgare were successfully expressed individually in Escherichia coli. The recombinant proteins were purified and used in enzyme assays. Further, the stability of the proteins at different temperatures and in different buffer solutions with various pH conditions was also tested. Protein crystals of R. capsulatus BchI were successfully formed in hanging drops by using 16% polyethylene glycol solution. Magnesium chelatase assays were performed at multiple timelines, temperature gradients and buffers at different pH. Furthermore, magnesium chelatase activity was tested with different combinations of recombinant R. capsulatus and H. vulgare protein subunits in combination with barley chloroplast extract. It was observed that recombinant R. capsulatus protein subunits did not influence the formation of Mg-deuteroporphyrin IX when combined with chloroplast extract whereas addition of recombinant H. vulgare protein subunits stimulated activity. It was also found that recombinant H. vulgare protein subunits showed magnesium chelatase activity by themselves. However, increasing amounts of added recombinant Xantha-G protein subunit decreased the activity.}},
author = {{Mahdi, Rabab}},
language = {{eng}},
note = {{Student Paper}},
title = {{Characterization of Rhodobacter capsulatus and barley (Hordeum vulgare L.) magnesium chelatases}},
year = {{2019}},
}