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

Morris , James C

Committee Member

Marcotte , William R

Committee Member

Paul , Kimberly S


Acetyl-CoA, an essential metabolite at the junction of various anabolic and catabolic pathways, is generated both from the breakdown of carbohydrates, lipids, and amino acids, and from the activation of acetate. AMP-forming acetyl-CoA synthetase, a key enzyme for acetate activation into acetyl-CoA in all domains of life, is a member of the adenylate-forming enzyme superfamily. Although members in the superfamily have different biological functions, they all share the property of forming an acyl-adenylate intermediate. Here I describe my research on characterization of a medium-chain acyl-CoA synthetase from Methanosarcina acetivorans (MacsMa) and identification of key residues in acyl substrate and CoA binding and catalysis. Additionally, this dissertation reports the characterization of Acs4 from Methanosaeta concilii (Acs4Msc) and further investigation of the role of a highly conserved Trp residue in Acs.
MacsMa was biochemically characterized to catalyze the acyl-CoA synthesis with preference for 2-methylbutyrate. However, when propionate was used as the acyl substrate, propionyl-CoA was not produced. Propionyl-AMP and PPi were released in the absence of CoA, whereas in the presence of CoA, the propionyl-AMP intermediate was converted to AMP and propionate and released along with PPi. These findings suggested that although acyl-CoA synthetases may have the ability to utilize a broad range of substrate for the acyl-adenylate forming first step of the reaction, the intermediate may not be suitable for the thioester-forming second step, in which case the enzyme has devised a mechanism for the release of the propionyl-adenylate intermediate.
In order to investigate the molecular basis of enzymatic mechanism of MacsMa, several key amino acid residues were identified through inspecting the enzyme structure and sequence alignment. Kinetic characterization of these residues suggested that MacsMa has highly conserved acyl and CoA binding pockets. Six residues, Lys256, Cys298, Gly351, Trp259, Trp237, and Trp254 were identified to be essential in acyl substrate binding and catalysis and also Lys256 had an important role in structure stability; Seven residues, Lys519, Lys461, Gly459, Tyr460, Arg490, Tyr525, and Tyr527 were found to be important in CoA binding and catalysis without significantly affecting the first half adenylate-forming reaction. Another interesting finding was that 3'-phosphate of CoA was proved to be critical in catalysis of thioester-forming step of the reaction in MacsMa. However, Acs is able to utilize 3'-dephospho CoA well though not as efficient as with CoA. Characterization of CoA binding and catalysis also suggested that MacsMa had a similar CoA binding pocket to CBAL.
Investigation of the MacsMa and active site(s) help gain a better understanding of biochemistry and two-step reaction mechanism in Acs and the evolution of the active site and substrate binding pocket in short- and medium-chain acyl-CoA synthetase (Sacs/Macs) family.
In order to investigate the biochemistry and kinetics of the Methanosaeta Acs's, Acs4Msc was biochemically characterized to show little to no acyl-adenylate synthetase/acyl-CoA synthetase activity. The Trp416 in Methanothermobacter thermautotrophicus Acs1 (Acs1Mt) has been shown to be highly conserved in other Acs's and very important in determining acyl substrate utilization but replaced by Phe in Acs4Msc. To further investigate the role of this highly conserved Trp active site in Acs, the Phe528Trp Acs4Msc variant was characterized to still display little to no acyl-adenylate/acyl-CoA synthetase activity as observed with wild-type enzyme. Furthermore, the Acs1Mt Trp416Phe variant showed similar kinetic parameters for either acetate or propionate for acyl-CoA synthetase; however, this variant released propionyl-adenylate as a free product, suggesting that Acs1Mt have different interactions with acetate and propionate and the benzoyl ring in Phe may reduce the ability of the enzyme to retain the propionyl-adenylate for the second-half thioester-forming step.
The appendix section includes a recently published paper focusing on the structure of MacsMa. I contributed to the enzymatic activity characterization of MacsMa and am an author on this paper.

Included in

Biochemistry Commons



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