In vivo cofactor biosynthesis and maintenance in the class Ia ribonucleotide reductase small subunit of Escherichia coli
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2009.
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| Format: | Thesis |
| Language: | eng |
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Massachusetts Institute of Technology
2009
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| Online Access: | http://hdl.handle.net/1721.1/46385 |
| _version_ | 1811072270917959680 |
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| author | Wu, Chia-Hung, Ph. D. Massachusetts Institute of Technology |
| author2 | JoAnne Stubbe. |
| author_facet | JoAnne Stubbe. Wu, Chia-Hung, Ph. D. Massachusetts Institute of Technology |
| author_sort | Wu, Chia-Hung, Ph. D. Massachusetts Institute of Technology |
| collection | MIT |
| description | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2009. |
| first_indexed | 2024-09-23T09:03:16Z |
| format | Thesis |
| id | mit-1721.1/46385 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T09:03:16Z |
| publishDate | 2009 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/463852019-04-10T20:28:47Z In vivo cofactor biosynthesis and maintenance in the class Ia ribonucleotide reductase small subunit of Escherichia coli Wu, Chia-Hung, Ph. D. Massachusetts Institute of Technology JoAnne Stubbe. Massachusetts Institute of Technology. Dept. of Chemistry. Massachusetts Institute of Technology. Dept. of Chemistry. Chemistry. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2009. This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. Vita. Includes bibliographical references. The small subunit ([beta]2) of Escherichia coli class Ia ribonucleotide reductases (RNRs) contains a diferric tyrosyl radical (Y*) cofactor essential for the conversion of nucleotides to deoxynucleotides that are needed for DNA synthesis and repair. The mechanism and factors involved in the biosynthesis, maintenance and regulation of this cluster remains unclear. To understand these pathways, the genes contiguous to nrdB (gene encoding [beta]) in 181 bacterial genomes were analyzed which revealed a highly conserved [2Fe2S]-ferredoxin, YfaE in E. coli. YfaE has been cloned, expressed, reconstituted, and characterized by UV-visible, EPR and Mössbauer spectroscopies. Titration of met-[beta]2 (an inactive diferric-[beta]2 with Y* reduced) with [2Fe2S]1+-YfaE results in formation of diferrous-[beta]2 with one Fe reduced/YfaE oxidized. At the end point of titration, exposure of the reduced cluster to O2 in the absence of an additional reducing equivalent yields the diferric-Y* with 2 Fe/Y* generated, suggesting that the reducing equivalent required for cluster assembly is supplied by [beta]2, likely by W48. The kobs for the reaction between met-[beta]2 and [2Fe2S]1+-YfaE determined by anaerobic stopped flow spectroscopy is ~1-5 s-1. Studies of conserved Lys to Ala mutations of [beta]2 indicate electrostatic interactions may play an important role for interaction with YfaE. Quantitative Western blots of the whole cells suggest that YfaE acts catalytically in reactivating met-[beta]2 in vivo. Titration experiments establish that met-[beta]2 can be reduced by catalytic amounts of YfaE, Fre (a flavin reductase) and flavin with consumption of NADPH. In the presence of a Y* scavenger, hydroxyurea, [delta]yfaE shows slower growth rates than the isogenic wt strain and Western blots analysis shows up-regulation of YfaE expression, supporting YfaE's role in the reactivation of diferric-[beta]2 in vivo. To investigate the iron sources for diferric-Y* assembly, changes in Fe pools inside the cell subsequent to expression of [beta]2 was monitored by whole cell Mössbauer spectroscopy. The results show that both Fe2+ and Fe3+ pools can provide the iron for cluster assembly, suggesting a reduction mechanism(s) for Fe3+ to allow it function in this capacity. A potential role of YfaE as an iron chaperone for iron delivery to [beta]2 has also been investigated. by Chia-Hung Wu. Ph.D. 2009-08-25T18:01:18Z 2009-08-25T18:01:18Z 2009 2009 Thesis http://hdl.handle.net/1721.1/46385 426940388 eng M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. http://dspace.mit.edu/handle/1721.1/7582 362 p. application/pdf Massachusetts Institute of Technology |
| spellingShingle | Chemistry. Wu, Chia-Hung, Ph. D. Massachusetts Institute of Technology In vivo cofactor biosynthesis and maintenance in the class Ia ribonucleotide reductase small subunit of Escherichia coli |
| title | In vivo cofactor biosynthesis and maintenance in the class Ia ribonucleotide reductase small subunit of Escherichia coli |
| title_full | In vivo cofactor biosynthesis and maintenance in the class Ia ribonucleotide reductase small subunit of Escherichia coli |
| title_fullStr | In vivo cofactor biosynthesis and maintenance in the class Ia ribonucleotide reductase small subunit of Escherichia coli |
| title_full_unstemmed | In vivo cofactor biosynthesis and maintenance in the class Ia ribonucleotide reductase small subunit of Escherichia coli |
| title_short | In vivo cofactor biosynthesis and maintenance in the class Ia ribonucleotide reductase small subunit of Escherichia coli |
| title_sort | in vivo cofactor biosynthesis and maintenance in the class ia ribonucleotide reductase small subunit of escherichia coli |
| topic | Chemistry. |
| url | http://hdl.handle.net/1721.1/46385 |
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