Structural investigations of class la ribonucleotide reductases by electron microscopy

Ribonucleotide reductase (RNR) catalyzes the reduction of nucleotides to their 2'-deoxynucleotide counterparts. The class la RNR from Escherichia coli is composed of two homodimeric subunits [alpha]2 and [beta]2 that form an [alpha]2[beta]2 complex to perform nucleotide reduction. Chemistry is initiated by a thiyl-radical (C439·) in the active site of [beta]2 that is reversibly generated by a diferric-tyrosyl radical cofactor (Y122·) in [beta]2 by a series of proton-coupled electron transfer steps: Y122[beta] <-> [W48[beta]] <-> Y356[beta] <-> Y731[alpha] - Y730[alpha] - C439[alpha]. A high-resolution structure of the active [alpha]2[beta]2 complex has long eluded the field due to the weak and transient nature of the a2-P2 interaction. Previous studies revealed that perturbing radical transfer by incorporating unnatural amino acids along the transfer pathway, or by using mechanistic inhibitors that trap the radical in the active site, can extend the lifetime of the [alpha]2[beta]2 complex, allowing for structural studies. Here, we present our efforts to study the E. coli class la RNR [alpha]2[beta]2 complex, trapped using these different perturbation methods, using cryo-electron microscopy. The two [alpha]2[beta]2 structures presented here provide deeper insight into the structural dynamics of nucleotide reduction. We end with a brief discussion of class la RNR from T4 bacteriophage, which despite sharing high sequence identity to its host E. coli class la RNR, employs a very different mode of oligomeric regulation.

Name thesis:degree_name

Doctoral

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Chemistry

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2019

Author dc:creator

• Kang, Gyunghoon.

Advisor dc:contributor.advisor

• Catherine L. Drennan.

• Chemistry.

Statement dc:rights

• MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission.

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/1721.1/123568

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/123568