| dc.description.abstract | Methane produced from anaerobic digestion of organic wastes has great potential as an alternative to fossil fuels. Anaerobic digestion combines the production of renewable energy with environmental benefits, including the reduction of greenhouse emissions, controlled waste disposal, and recycling of nutrients. Biomethanation or anaerobic digestion relies on fermenting bacteria, syntrophs, and methanogens working synergistically to break down large organic molecules to produce methane and carbon dioxide. Within this process, syntrophic metabolism is the rate-limiting step due to its low Gibbs free energy change and the reliance on methanogens to degrade the syntrophic by-products. Therefore, methane production requires efficient metabolic coordination of syntrophic and methanogenic organisms in energetically limiting conditions. While much is known about the physiology and metabolism of methanogens, large knowledge gaps remain surrounding the metabolic processes of syntrophic microorganisms due to the difficulty of cultivation and growth in pure culture. A deeper understanding of the enzymes and metabolic mechanisms utilized by model syntrophs will inherently allow for an increase in the controlled biological production of methane, which can then be used as a source of energy. In this dissertation, two vital mechanisms involved in energy conservation and electron transport within the model syntrophic organisms, Syntrophus aciditrophicus strain SB and Syntrophomonas wolfei were characterized.
Syntrophus aciditrophicus strain SB is a model syntroph and is representative of syntrophic bacteria that have an essential role, along with fermentative and methanogenic organisms, in the anaerobic recycling of organic matter to methane and carbon dioxide. Previous studies showed that S. aciditrophicus shares a core set of reversible enzymes for the benzoate and cyclohexane-1-carboxylate metabolic pathways. Within these metabolic pathways, S. aciditrophicus utilizes a novel mechanism to synthesize ATP via an AMP-forming acetyl-CoA synthetase (SaAcs1). AMP-forming acetyl-CoA synthetases (Acs) typically synthesize acetyl-CoA and AMP from acetate and ATP in two consecutive steps: an adenylate-forming step and a thioester-forming step. However, SaAcs1 favors the reverse reaction, resulting in ATP synthesis. One goal of this dissertation was to structurally characterize SaAcs1 to investigate the role that the structure of the enzyme has on the function, as well as functionally characterize the enzyme using kinetics alongside the exploration of the role of specific amino acids within the enzyme that allow it to favor ATP production.
A 2.2 Å resolution structure of SaAcs1WT bound to acetyl-AMP in the adenylation conformation was resolved using X-ray crystallography. The structural alignment of SaAcs1WT with previously described Acs-family proteins revealed key structural differences from other known members of the protein family. The noted structural differences in SaAcs1 provide insight into the molecular mechanisms that may bias it to operate in the reverse, less thermodynamically favorable direction. The primary structural difference involved in the repositioning of a well-conserved loop region in the CoA substrate binding pocket. The structure of SaAcs1WT in the adenylation conformation contributes to the body of research on Acs-family proteins. Currently, in the Protein Data Bank (PDB), only two organisms have published structures of the Acs protein in the adenylation conformation, while there are over a dozen published Acs structures in the alternate or thioester-forming conformation. Additionally, enzyme activity and kinetics assays performed on SaAcs1 confirmed SaAcs1 favorably functions in the ATP-forming reaction compared to other Acs enzymes, which favor the typical AMP-forming reaction. Additionally, this work is the first to compare the activity of Acs-family proteins in the ATP-forming direction.
To determine whether specific amino acid residues are important for SaAcs1 to function in the ATP-forming direction, site-directed mutagenesis was utilized to create SaAcs1 variants with single amino acid substitutions into residues hypothesized to be critical to function. The variants were grouped into four categories based on the region of the targeted mutations: at the beginning/within the CoA loop, the conserved residues within the CoA loop, within the ATP-binding pocket, and at the hinge position. Four structures of the SaAcs1 variants were also resolved using X-ray crystallography and the kinetic activity of all variants was measured in the ATP-forming and AMP-forming directions. These substitutions either resulted in no change of activity from the wild-type or a change of activity – increased activity, decreased activity, or no enzyme activity. Both structural and kinetic data of the SaAcs1 variants confirmed that the loop next to the CoA binding pocket of SaAcs1WT impacts the function of the enzyme to favor the ATP-forming reaction.
Syntrophomonas wolfei serves as a model for the syntrophic metabolism of the short-chain fatty acids such as butyrate to the methanogenic substrates: hydrogen, formate, and acetate. One important but poorly understood process in syntrophic metabolism is the generation of electrons from the oxidation of acyl-CoA intermediates coupled to the production of hydrogen or formate or the process of reverse electron transfer. The S. wolfei genome has three gene clusters encoding for Etf, all of which accept electrons from an acyl-CoA dehydrogenase. The structure of electron transfer flavoprotein (EtfAB3) from Syntrophomonas wolfei was resolved to 2.5 Å with a bound novel ADP ligand and FAD. The genes encoding for the SwEtfAB3 complex are associated with the Fix complex genes and were proposed to have a role in electron transfer during β-oxidation. The structural characterization of SwEtfAB3 contributes to understanding the unique metabolic machinery of syntrophic metabolizers and their ability to produce hydrogen or formate from high redox potential electrons.
In this dissertation, the key mechanisms to support the syntrophic metabolism of S. aciditrophicus and S. wolfei were investigated to understand the complex and dynamic microbial processes associated the ability of syntrophs to exploit small free energy changes and the systems involved in electron flow. The primary objective of this dissertation was to provide additional information on novel ATP synthesis from Syntrophus aciditrophicus (Sa) strain SB. Additionally, the characterization of core genes involved in reverse electron transport from S. wolfei provides additional information on the proposed mechanisms involved in syntrophic metabolism. Although some research has been conducted to study metabolic syntrophy in anaerobic environments, there is still missing information on how energy is conserved in syntrophic metabolism. An increasingly demanding research field involves engineering systems to improve controlled biological production of methane and thus increase energy production. With these pursuits, a fundamental understanding of syntrophic metabolism is necessary to develop novel systems that improve small and large-scale biomethanation. | en_US |
