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Catherine L. Drennan. Jonnalagadda, Rohan. Massachusetts Institute of Technology. Department of Biology. Massachusetts Institute of Technology. Department of Biology 2020-09-03T17:48:22Z 2020-09-03T17:48:22Z 2020 2020 https://hdl.handle.net/1721.1/127130 1191837325 Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, May, 2020 Cataloged from the official PDF of thesis. Vita. Includes bibliographical references. 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. by Rohan Jonnalagadda. Ph.D. Ph.D. Massachusetts Institute of Technology, Department of Biology 162 pages eng Massachusetts Institute of Technology 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. http://dspace.mit.edu/handle/1721.1/7582 Biology. Structural and functional investigations of mechanisms of iron-utilizing enzymes Thesis application/pdf 2020-09-03T17:48:22Z Publication Doctoral Bio unknown unknown <Publication xmlns="https://www.openaire.eu/cerif-profile/1.1/" id="e08e752f-14e7-4538-bf95-85155bf5299e"> <Type xmlns="https://www.openaire.eu/cerif-profile/vocab/COAR_Publication_Types">http://purl.org/coar/resource_type/c_1843</Type> <Language>eng</Language> <Title>Structural and functional investigations of mechanisms of iron-utilizing enzymes</Title> <PublishedIn> <Publication> </Publication> </PublishedIn> <PublicationDate>2020</PublicationDate> <Authors> <Author> <DisplayName>Jonnalagadda, Rohan.</DisplayName> <Affiliation> <OrgUnit> </OrgUnit> </Affiliation> </Author> </Authors> <Editors> </Editors> <Publishers> <Publisher> <DisplayName>Massachusetts Institute of Technology</DisplayName> <OrgUnit /> </Publisher> </Publishers> <License>http://dspace.mit.edu/handle/1721.1/7582</License> <Keyword>Biology.</Keyword> <Abstract>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.</Abstract> <Access xmlns="http://purl.org/coar/access_right" > </Access> </Publication>