Date of Award


Degree Type


Degree Name

Doctor of Philosophy


Life Sciences

Major Professor

Elizabeth E. Howell, Engin H. Serpersu

Committee Members

Gladys M. Alexandre-Jouline, Jerome Baudry, Daniel M. Roberts


Bacteria can acquire resistance against antibiotics by covalently modifying them. This is achieved by plasmid-encoded enzymes called as aminoglycoside modifying enzymes (AGMEs). More than 50 different AGMEs are known currently, having variable levels of substrate promiscuity. Even though these enzymes are highly similar on the sequence level and also share a similar structural fold, the molecular basis of promiscuity is not clearly understood. We aim to characterize enzymes with high and low substrate promiscuity and compare their properties to better understand ligand selectivity of AGMEs.This project describes the kinetic, thermodynamic and structural properties of aminoglycoside N3 acetyltransferase VIa (AAC-VIa). It is one of the least promiscuous AGMEs, being able to modify only five aminoglycosides. Unlike the more promiscuous aminoglycoside N3-acetyltransferase IIIb (AAC-IIIb), AAC-VIa can kinetically distinguish between its various substrates. Thermodynamic studies determined the binding of ligands to be enthalpically driven and entropically unfavorable. Unlike other AGMEs, the formation of binary and ternary complexes was accompanied by a net deprotonation of the enzyme, ligand or both. Analytical ultracentrifugation (AUC) studies showed that AAC-VIa exists in a monomer-dimer equilibrium, with more dimeric form appearing with increasing concentrations of the enzyme. Binding of ligands drive the enzyme to the monomeric form. Also, dimer formation, unlike the AAC-IIIb, was observed to be achieved through polar interactions. X-ray crystallography using the apo- and ligand bound forms of AAC-VIa was performed to decipher the catalytic mechanism of acetylation. Crystal structures of different complexes of the enzyme showed that structures of apo- and ligand-bound forms are similar which suggests that, unlike other AGMEs, more rigid structure of AAC-VIa may limit the active site to accommodate only few selected aminoglycosides, hence low substrate promiscuity. The structures also suggested a novel catalytic mechanism involving a non-classical catalytic triad. Neutron diffraction studies were used to identify a low barrier hydrogen bond between the active site residues. Overall, we highlight the contrasting properties of the high and low substrate promiscuous acetyltransferases, while also being the first one to report the unique mechanism of acetyl transfer. Results from this work can aid the development of next generation of aminoglycoside drugs.

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