Structural and biochemical investigations into the Neisseria gonorrhoeae ribonucleotide reductase

Abstract

Ribonucleotide reductase (RNR) is the only known enzyme that converts ribonucleotide substrates to their deoxyribonucleotide products necessary for DNA synthesis and repair. For class Ia RNRs, this chemically-difficult process is tightly regulated at many levels including through allosteric regulation, which occurs in many organisms through oligomeric state changes. These quaternary shifts allow for radical transfer to the active site in the presence of allosteric activator ATP (active state) and prevent that radical transfer in the presence of allosteric inhibitor dATP (inactive state).

The essential reaction that RNR catalyzes, along with its distinct active and inactive states, makes RNR both a current anticancer drug target and a promising antibiotic target. Differences in composition of inactive states between human and some bacterial RNRs further elevates RNR as a potential selective antibiotic target. This thesis structurally and biochemically characterizes the ribonucleotide reductase enzyme from Neisseria gonorrhoeae, the causative agent of gonorrhea. N. gonorrhoeae is a prevalent public health concern presenting a current and imminent risk for multi- and fully-drug resistant strains. Here, we biochemically characterize and present an inactive structure of the N. gonorrhoeae RNR, which is the first structure of an RNR from a pathogenic organism. We present and characterize compounds that selectively inhibit the N. gonorrhoeae RNR in vitro and in vivo. We also present an LC-MS/MS assay for use characterizing the activity of a diverse array of RNRs under different nucleotide conditions.

This thesis additionally explores the use of the chameleon, an automated cryo-electron (cryo-EM) specimen preparation instrument, in generating cryo-EM specimens of the N. gonorrhoeae RNR suitable for high-resolution reconstructions, as traditional blot-based plunging techniques were unable to yield intact particles for reconstruction. General optimization of conditions for use of the chameleon for difficult cryo-EM samples is discussed along with presentation of the Neisseria gonorrhoeae inactive RNR structure as a case study. This work will open the door for future research on the N. gonorrhoeae RNR as a potential antibiotic target and for use of the chameleon for generation of high resolution reconstruction for a broad range of difficult cryo-EM samples.