Graduate Studies

 

First Advisor

Toshihiro Obata

Degree Name

Doctor of Philosophy (Ph.D.)

Committee Members

Donald becker, Limei Zhang, Oleh Khalimonchuk, Rajib Saha

Department

Biochemistry

Date of this Version

8-2024

Document Type

Dissertation

Citation

A dissertation presented to the faculty of the Graduate College of the University of Nebraska in partial fulfillment of requirements for the degree of Doctor of Philosophy

Major: Biochemistry

Under the supervision of Professor Toshihiro Obata

Lincoln, Nebraska, August 2024

Comments

Copyright 2024, Joy John Omini. Used by permission

Abstract

The TCA cycle enzymes- malate dehydrogenase (MDH) and citrate synthase (CS)- interact to transfer oxaloacetate from the active site of MDH to the active site of CS, through an electropositive channel. The MDH-CS metabolon is highly conserved across different domains of life, and it has been associated with many advantages. Some of these advantages include protecting oxaloacetate from competing enzymes like aspartate aminotransferase, allowing the thermodynamically unfavorable MDH forward reaction to occur and increasing the local concentration of oxaloacetate within the active site of CS. These advantages and conservativeness of the MDH-CS metabolon indicate that it is highly important to the cell, hence the aim of this study is to discover the function of the MDH-CS metabolon in living cells.

The MDH-CS metabolon was studied in vitro and in living yeast cells under various metabolic conditions including fermentative, respiratory and respiro-fermentative growth conditions. The MDH-CS metabolon dynamically interacted in vitro and in vivo, depending on the microenvironment and available substrates. Substrates of the MDH and CS reactions like malate and acetyl CoA enhanced association of the metabolon while products like citrate caused dissociation. Other factors like the presence of reducing equivalents, ATP and the pH of the microenvironment altered MDH-CS metabolon interaction in vitro. In living yeast cells specifically, the MDH-CS metabolon association was enhanced when respiration was induced, and dissociation was enhanced at the onset of the Crabtree effect. Also, acidic and oxidizing mitochondria matrix favored assembly of the MDH-CS metabolon while reducing matrix favored its dissociation. Disruption of the MDH-CS metabolon by mutation of yeast mitochondrial citrate synthase decreased MDH and CS fluxes, increased aspartate aminotransferase flux and altered metabolite accumulation in respiro-fermentative and respiratory conditions. Interestingly, dissociation of the MDH-CS metabolon decreased overall TCA cycle flux only in fully respiratory conditions while the metabolism of cells grown on fermentable carbon source was unaffected by the dissociation of the metabolon.

The results of this thesis study show that the MDH-CS metabolon is a dynamically interacting regulatory machinery utilized by living cells to regulate TCA cycle flux in accordance with the metabolic need of the cell.

Advisor: Toshihiro Obata

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