Home WakeSpace Scholarship › Electronic Theses and Dissertations

EVALUATION OF SULFUR TRANSFER REACTIONS REQUIRED FOR THE SYNTHESIS OF COFACTORS IN BACTERIAL SYSTEMS

Electronic Theses and Dissertations

Item Files

Item Details

title
EVALUATION OF SULFUR TRANSFER REACTIONS REQUIRED FOR THE SYNTHESIS OF COFACTORS IN BACTERIAL SYSTEMS
author
Addo, Maame Adwoa Amponsah
abstract
The amino acid cysteine serves as the sulfur source for the biosynthesis of thiocofactors. Cysteine desulfurases are PLP-containing enzymes involved in the initial step of sulfur activation and trafficking from cysteine to biomolecules such as nucleosides, Fe-S clusters, and vitamins. The study of sulfur trafficking for the synthesis of these cofactors is challenging because their associated biosynthetic schemes often include shared reaction intermediates and sulfur acceptor proteins participating in multiple pathways. Notably, some pathways require the participation of Fe-S enzymes, and defects in Fe-S cluster biogenesis indirectly affect the synthesis of such cofactors. In addition to being involved in the synthesis of thiocofactors, sulfur trafficking pathways have also been shown to affect the synthesis of non-thiolated metabolites through mechanisms that are not fully explored. This study has expanded the understanding of pathways involving sulfur transfer reactions through investigation of the occurrence of biosynthetic components across prokaryotic genomes, characterization of sulfur transfer reactions directing the synthesis of Fe-S clusters in nitrogen fixation, and the involvement of sulfur transfer reactions on non-thiolated tRNA nucleosides.This dissertation surveys the distribution of nitrogen fixation genes in diazotrophic prokaryotes known or proposed to contain alternative nitrogenase systems. This study not only expands the inventory of diazotrophs and solidifies the minimum group of genes (nifHDKENB) required for diazotrophy, but also establishes the likelihood of NifUS function in sustaining the high demand for Fe-S clusters during aerobic nitrogen fixation. Furthermore, this work focuses on understanding the individual steps of sulfur transfer and reduction during the assembly of Fe-S clusters by A. vinelandii NifS and NifU. We have demonstrated that the host organism and demand for nitrogen fixation affect the cluster occupancy of NifU and FPR2 is a suitable physiological reductant in the synthesis of Fe-S clusters on NifU. This study reports the involvement of sulfur transfer reaction in the synthesis and non-canonical functions of tRNA modifications. The levels of these decorations on tRNA molecules fluctuate depending on the stressor (environmental or nutrient availability) and can be used as a method to assess cellular status. Additionally, experimental investigation has furthered our understanding of the link between the Csd sulfur trafficking pathway and the synthesis of a non-thiolated metabolite, ct6A tRNA. We proposed a mechanism for ct6A formation that includes the formation of a thioester intermediate following adenylation. The occurrence of this intermediate rapidly induces cyclization to form ct6A, and the involvement of artificial alkyl thiols (e.g. DTT and BME) masks the involvement of physiological sulfur-intermediates assisting catalysis in vivo. Collectively, this work expands the scope of biochemical reactions involving the synthesis of cofactors that recruit the participation of cysteine desulfurases and sulfur tracking protein partners. This dissertation reports on new and previously-not-understood aspects of sulfur-dependent and independent pathways relevant in microbial systems.
subject
alanine
cysteine
dithiothreitol
iron-sulfur
liquid chromatography mass spectrometry
serine
contributor
Dos Santos, Patricia C (advisor)
Alexander, Rebecca W (committee member)
King, Bruce (committee member)
Stich, Troy A (committee member)
date
2023-01-24T09:35:55Z (accessioned)
2022 (issued)
degree
Chemistry (discipline)
embargo
2027-12-31 (terms)
2027-12-31 (liftdate)
identifier
http://hdl.handle.net/10339/101783 (uri)
language
en (iso)
publisher
Wake Forest University
type
Dissertation

Usage Statistics