Date of Award

5-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Biochemistry and Molecular Biology

Advisor

Kerry S Smith

Committee Member

William Marcotte

Committee Member

James Morris

Committee Member

Meredith Morris

Committee Member

Lukasz Kozubowski

Abstract

Although acetate is a predominant metabolite produced by many eukaryotic microbes, far less attention has been given to acetate metabolism in eukaryotes than in bacteria and archaea. Acetate kinase (Ack), which catalyzes the reversible phosphorylation of acetate from ATP, is a key enzyme in bacterial acetate metabolism. Ack primarily partners with phosphotransacetylase (Pta), which catalyzes the generation of acetyl phosphate from acetyl-CoA, but can also partner with xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), which produces acetyl phosphate from either xylulose 5-phosphate or fructose 6-phosphate. The Ack-Pta pathway, found primarily in bacteria, is also present in lower eukaryotes such as the green algae Chlamydomonas reinhardtii and the oomycete, Phytophthora. The Ack-Xfp pathway, which forms a modified pentose phosphoketolase pathway in heterofermentative bacteria, has been found in a number of ascomycete and basidiomycete fungi. Although bacterial and eukaryotic microbes possess these pathways, humans, animals and plants lack these enzymes, making this pathway a potential drug target in eukaryotic pathogens. Two types of Ptas have previously been identified: PtaI and PtaII. PtaII enzymes have an N-terminal regulatory domain that the PtaI enzymes lack. Through sequence analysis, we identified four subtypes, IIa, IIb, IIc, and IId, of the PtaII enzymes based on the presence or absence of two N-terminal subdomains. Here we describe the first biochemical characterization of a eukaryotic Pta, the Phytophthora ramorum Type IIa Pta1 (PrPta1IIa). Although the N-terminus of PrPta1IIa shares only 19% amino acid identity with the N-terminus of the bacterial Escherichia coli and Salmonella enterica PtaIIa enzymes, the effector molecules, ATP, NADH, PEP, and pyruvate, inhibit all three enzymes in the acetyl-CoA-forming direction; whereas, AMP differentially regulates PrPta1IIa compared to SePtaIIa. We hypothesize that Xfp-Ack would function as a modified pentose phosphoketolase pathway to produce acetate and ATP in the opportunistic, fungal pathogen Cryptococcus neoformans, which has two open reading frames, designated as Xfp1 and Xfp2, with sequence identity to Xfp. To investigate the metabolic and physiological role of the Ack-Xfp pathway in C. neoformans, we have generated single XFP1, XFP2 and ACK knockouts, as well as a XFP1/XFP2 double knockout. Our results indicate both Xfp1 and Xfp2 play a role in the survival of C. neoformans within macrophages, and that Ack and Xfp2 most likely partner together under low glucose and possibly low iron environments.

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