Investigations of conserved structure-function relationships and enzymatic evolution

dc.contributorDunkle, Jack A.
dc.contributorJenny, Matthew J.
dc.contributorPapish, Elizabeth T.
dc.contributorWoski, Stephen A.
dc.contributor.advisorFrantom, Patrick A.
dc.contributor.authorConte, Juliana Victoria
dc.contributor.otherUniversity of Alabama Tuscaloosa
dc.date.accessioned2021-07-07T14:36:53Z
dc.date.available2021-07-07T14:36:53Z
dc.date.issued2021
dc.descriptionElectronic Thesis or Dissertationen_US
dc.description.abstractThe E.coli SUF pathway is the iron-sulfur cluster biogenesis pathway responsible for assembling iron-sulfur clusters during times of oxidative stress and iron starvation. SufS is the cysteine desulfurase enzyme responsible for the acquisition of sulfur and subsequent transfer to transpersulfurase SufE. Recent studies have provided several crystal structures of SufS including two variants displaying stalled intermediates of the desulfurase reaction. Combining the structural data with biochemical investigations has uncovered several key elements in SufS activity. SufS is a homodimer proposed to use a half-sites mechanism involving conformational changes and communication through dimer interface interactions. Despite the significant contributions made to understanding SufS function, questions still remain about the specific roles of several structural elements involved in activity and regulation. Investigations discussed herein are aimed at probing structural components of SufS, including the beta hairpin structure. The beta hairpin makes up one wall of the active site of the opposite monomer. The proposed regulatory mechanism of SufS uses the beta hairpin dynamics to control access to the active site. In chapter 2 beta hairpin variants are shown to significantly disrupt SufS structure. Variants were unable to bind the PLP cofactor essential to catalysis and instability of the variants resulted in protein aggregation as demonstrated with size exclusion chromatography analysis. Chapter 3 focuses on single substitutions of residues located in and around the active site including Asn99, Arg56, and Arg359. Kinetic assays were conducted to determine defects in desulfurase activity by measuring the rate of alanine production, the product released by the desulfurase reaction. Asn99 variants exhibited a ten-fold decrease in turnover number, confirming Asn99 does play a role in generating an optimal environment for activity. This is likely due to the hydrogen bonds Asn99 forms with the glycine residues in the loop at the base of the beta hairpin, potentially contributing to the regulation of dynamics. Arg56 is located in a dynamic loop above the active site and was previously proposed to contribute to catalysis by hydrogen bonding with the sulfhydryl of the substrate cysteine. Kinetic analysis of both R56A and R56K variants is consistent with Arg56 having a significant role in SufS activity. It is possible that this residue is responsible for the deprotonation of SufE C51 and facilitating persulfide transfer. Kinetic analysis of the Arg359 variants suggest a role in cysteine binding and positioning the substrate for bond cleavage. The work presented here contributes to the current understanding of the SufS mechanism and may be more broadly applicable to other cysteine desulfurases.en_US
dc.format.extent130 p.
dc.format.mediumelectronic
dc.format.mimetypeapplication/pdf
dc.identifier.otheru0015_0000001_0003791
dc.identifier.otherConte_alatus_0004D_14396
dc.identifier.urihttp://ir.ua.edu/handle/123456789/7870
dc.languageEnglish
dc.language.isoen_US
dc.publisherUniversity of Alabama Libraries
dc.relation.hasversionborn digital
dc.relation.ispartofThe University of Alabama Electronic Theses and Dissertations
dc.relation.ispartofThe University of Alabama Libraries Digital Collections
dc.rightsAll rights reserved by the author unless otherwise indicated.en_US
dc.subjectBiochemistry
dc.titleInvestigations of conserved structure-function relationships and enzymatic evolutionen_US
dc.typethesis
dc.typetext
etdms.degree.departmentUniversity of Alabama. Department of Chemistry and Biochemistry
etdms.degree.disciplineChemistry
etdms.degree.grantorThe University of Alabama
etdms.degree.leveldoctoral
etdms.degree.namePh.D.
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