Author's School

Graduate School of Arts & Sciences

Author's Department/Program



English (en)

Date of Award

January 2009

Degree Type


Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Thomas Kappock


Acetobacter aceti oxidizes ethanol to acetic acid. While the membrane-permeable acetic acid is toxic to many bacteria, A. aceti survives exposure to acetic acid by tolerating cytoplasmic acidification. The ability to tolerate an acidic cytoplasm suggests that proteins from A. aceti are unusually suited to function in an acidic environment. The ability to tolerate an acidic cytoplasm raises additional questions about biosynthetic pathways that employ acid-labile intermediates. To examine how A. aceti metabolism may have adapted to function under acidic conditions, a biosynthetic conversion involving an acid-labile metabolite has been selected for study. The enzyme N5-carboxyaminoimidazole ribonucleotide mutase: PurE) catalyzes the reversible intramolecular transfer of the carboxylate of N5-carboxyaminoimidazole ribonucleotide: N5-CAIR) to the C-4 position of the imidazole ring to form 4-carboxyaminoimidazole ribonucleotide: CAIR). A series of mutants was made to explore the role of two conserved histidines. A pH-rate comparison of AaPurE and the active mutant AaPurE-H59D was used to identify His59 as the key active site acid/base residue. The thermostability of Escherichia coli PurE: EcPurE) over a range of pH was also assessed and compared to AaPurE. AaPurE was found to be significantly more thermostable than EcPurE over the entire pH range surveyed. Comparison of the pH-rate profiles constructed for AaPurE with recently reported pH-rate profiles for EcPurE indicate that the two do not differ significantly, indicating there has been no adaptive change in enzyme mechanism. Also reported is a summary and analysis of a number of crystal structures that have been determined for AaPurE, which suggests a strategy by which proteins may have become resistant to acid-mediated inactivation. Initial functional complementation studies using the purE auxotroph PC0135 suggested that AaPurE may require AaPurK to function. We constructed a set of stable E. coli deletion strains and insertion strains that replace the chromosomal copies of purEEc or purKEc with their counterparts from A. aceti. Functional complementation experiments suggest that a third protein, located upstream of purEAa and conserved in Rhodospirillales, may be involved in the proper functioning of AaPurE. The nonenzymatic decarboxylation of CAIR and the corresponding ribonucleoside: CAIR-s) were examined as models for the PurE reaction. The decarboxylation of CAIR-s was not acid-catalyzed in the pH range examined, and did not accelerate in lower-polarity solvents.


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