Structural and functional investigations of mechanisms of iron-utilizing enzymes

Biological systems use iron as a key cofactor to catalyze a variety of difficult chemical transformations, particularly as reservoirs from which to initiate enzymatic radical chemistry. Here, I discuss efforts to study the allosteric mechanisms of an essential human iron and free radical utilizing enzyme: ribonucleotide reductase (RNR), and efforts to elucidate the radical based mechanism of isonitrile formation by an mononuclear nonheme iron(II) α-ketoglutarate dependent dioxygenase, ScoE. The first part of this thesis focuses on mononuclear nonheme iron(II) α-ketoglutarate dependent dioxygenases: enzymes which use molecular oxygen, α-ketoglutarate and a mononuclear Fe(II) cofactor to initiate substrate radical chemistry and catalyze diverse chemistries such as hydroxylations, halogenations and desaturations. Chapter 1 of this thesis describes current understanding of the mechanisms of these enzymes. Chapter 2 describes biochemical and structural efforts to determine the catalytic mechanism of one member of these enzymes: the isonitrile formation enzyme ScoE. In the chapter 3 of this thesis, I discuss the structure, function and allosteric regulation of RNR, the sole known enzyme capable of catalyzing the de novo biosynthesis of deoxyribonucleotides in all organisms for use in DNA replication and repair. Chapter 4 concerns efforts to develop a procedure for the improved recombinant protein yield of human RNR, and chapter 5 describes the development of a new liquid chromatography-tandem mass spectrometry based assay for RNR activity that we propose holds promise for simultaneous detection of RNR activity on multiple substrates.

Name thesis:degree_name

Doctoral

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Biology

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2020

Author dc:creator

• Jonnalagadda, Rohan.

Advisor dc:contributor.advisor

• Catherine L. Drennan.

• Biology.

Statement dc:rights

• MIT theses may be protected by copyright. Please reuse MIT thesis content according to the MIT Libraries Permissions Policy, which is available through the URL provided.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1721.1/127130

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/127130