Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Biochemistry and Molecular Biology

Committee Chair/Advisor

Smith, Kerry S

Committee Member

Marcotte, William R

Committee Member

Morris, James C

Committee Member

Paul, Kimberly S


Acetate is excreted as a metabolic end product in many microbes. Acetate production has primarily been studied in bacteria and archaea but is known to occur in eukaryotic organisms as well. For example, acetate is one of the most abundant metabolites excreted by the fungal pathogen Cryptococcus neoformans during cryptococcosis suggesting that acetate production may be important during pathogenesis. One possible pathway for acetate production in C. neoformans involves the enzymes xylulose 5-phosphate/ fructose 6-phosphate phosphoketolase (Xfp), which can generate acetyl phosphate from either fructose 6-phosphate (F6P) or xylulose 5-phosphate (X5P), and acetate kinase (Ack), which can then convert acetyl phosphate to acetate. C. neoformans has an ACK and two XFP open reading frames that we've designated as XFP1 and XFP2, and several studies indicate that these transcripts are expressed and/or upregulated under infectious conditions. However, until now, the Xfp-Ack pathway in C. neoformans has not been studied. Here I describe the first characterization of a eukaryotic Xfp, the C. neoformans Xfp2. C. neoformans Xfp2 was found to display both substrate cooperativity and allosteric regulation. C. neoformans Xfp2 showed positive cooperativity in regards to F6P and X5P binding and negative cooperativity for Pi binding. Activity was inhibited by ATP, phosphenolpyruvate (PEP), oxaloacetic acid (OAA) and glyoxylate and activated by AMP. Both PEP and OAA were found to bind at the same or possess overlapping allosteric sites on the enzyme. Prior to this characterization of C. neoformans Xfp2 the only other phosphoketolases characterized have been from bacteria, and none report the presence of substrate cooperativity or allosteric regulation. Therefore, Lactobacillus plantarum Xfp was re-characterized to determine if the enzyme showed substrate cooperativity and/or allosteric regulation. L. plantarum Xfp displayed negative cooperativity for Pi binding and was also allosterically inhibited by PEP, OAA and glyoxylate, but activity was unaffected by the presence of AMP or ATP. Like C. neoformans Xfp2, PEP and OAA were found to share the same or overlapping allosteric sites on L. plantarum Xfp. This study proved that substrate cooperativity and allosteric regulation exist for at least some bacterial Xfps. Models of C. neoformans Xfp2 monomer and dimer were generated from existing bacterial Xfp crystal structures. Since bacterial and eukaryotic Xfps may share the PEP/OAA allosteric site, ligand docking simulations were performed with PEP and OAA in various proposed binding sites on the C. neoformans Xfp2 model. Site-directed mutagenesis was performed on residues predicted to hydrogen bond with PEP and OAA within these sites. However, these studies have yet to reveal the location of the PEP/OAA allosteric site. More recently the Xfp2 dimer model has revealed new sites formed between monomers that could serve as the PEP/OAA allosteric site.



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