Date of Award
Doctor of Philosophy (PhD)
Joseph T. Kappock
Joseph T. Kappock
Committee Member 1
Committee Member 2
Committee Member 3
Acetobacter aceti (A. aceti) is a Gram-negative, acidophilic bacterium that is used for the industrial production of acetic acid from ethanol. Oxidation of ethanol by membrane-bound oxidoreductases provides energy for A. aceti and the production of high concentrations of acetic acid is an effective defense mechanism. Acetic acid diffuses through cell membranes at low pH and effectively kills many bacteria, including E. coli, at low millimolar concentrations. The ability of A. aceti to thrive in molar concentrations of acetic acid is partially due to the twin subjects of this thesis, the acetic acid resistance factors AarA (citrate synthase, AaCS) and AarC (succinyl-CoA:acetate CoA-transferase).
AarC and CS exploit the distinct properties of the thioester moiety in acetyl-CoA (AcCoA) to catalyze different reversible reactions. AarC takes advantage of the relatively high leaving group potential of the CoA thiolate (anionic or RS– form) to transfer the acetyl moiety of AcCoA to an active site glutamate. In contrast, CS uses the relatively acidic carbon adjacent to the thioester moiety to catalyze a Claisen/aldol condensation reaction that forms a new carbon-carbon bond. Class I CoA-transferases such as AarC produce acylglutamyl anhydride intermediates that undergo attack by the CoA thiolate on one of its two carbonyl carbon atoms, forming distinct internal or external tetrahedral intermediates less than 3 Å apart. In this study, crystal structures were used to examine the role of the internal oxyanion hole residue Asn347 and the highly conserved elements of the external oxyanion hole.
First, a structure of the active mutant AarC-N347A bound to CoA revealed both solvent substitution for the deleted carboxamide and displacement of the adjacent Glu294. This indicates that Asn347 both polarizes and orients the essential glutamate Glu294.
Second, AarC was crystallized with the nonhydrolyzable AcCoA analogue dethiaacetyl-CoA (AcMX) in an attempt to trap a closed enzyme complex containing a stable analogue of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and a truncated AcMX, an unexpected result hinting at what would have been an unprecedented cleavage of the ketone moiety into an acetyl group and the CoA analogue MX. Solution studies confirmed that AcMX decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC.
Authentic MX was synthesized to evaluate the hypothesis that it is derived from AcMX. A crystal structure of AarC bound to MX showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even for crystals grown in the presence of exogenous acetate. These findings imply that AcMX degradation results in the production of an activated acetyl donor; a working hypothesis involving ketone oxidation is offered. Moreover, the ability of MX to induce full active site closure suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA. The remainder of this thesis concerns the enzyme CS, an essential part of central metabolism in aerobes and many other organisms. The CS reaction comprises two successive reactions: a Claisen/aldol condensation of AcCoA and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that close the active site assemble a catalytically competent condensation active site. The 2.2 Å resolution crystal structure of CS from the thermoacidophile Thermoplasma acidophilum (TpCS) fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an open structure that, when compared with several liganded TpCS structures, helps to define a complete path for active site closure. (Abstract shortened by ProQuest.)
Murphy, Jesse R., "Mechanistic characterization of acetic acid resistance enzymes of Acetobacer aceti" (2016). Open Access Dissertations. 688.