Date of Award

10-2003

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry

Supervisor

Professor G.D. Wright

Abstract

The clinical utility of aminoglycoside antibiotics is undermined by the action of bacterial modifying enzymes that inactivate these antimicrobial agents and confer drug resistance. Basic research on the aminoglycoside modifying enzymes has contributed to a greater understanding of the molecular mechanism of aminoglycoside detoxification by these proteins, information that is relevant to inhibitor studies and other approaches aimed at reversing bacterial aminoglycoside resistance in vivo. Described here is the characterization of AAC{6')-li, a chromosomally encoded aminoglycoside 6' -Nacetyltransferase from the Gram positive pathogen Enterococcus faecium and a member of the GCNS-related superfamily of acyltransferase enzymes. Research on AAC{6')-li has focused on the kinetic and catalytic mechanism of aminoglycoside modification by this enzyme, in addition to inhibitor studies and investigations into a possible alternate role for this acetyltransferase in vivo. Using steady state-kinetic analysis of product and dead-end inhibition of protein activity, we have determined that AAC{6')-li follows a ternary complex, ordered bi-bi kinetic mechanism, with additional evidence supporting subunit cooperativity in the AAC{6')-Ii dimer. This work was complemented by studies that characterized substrate-induced conformational changes in AAC{6')-Ii, as well as analysis of solvent viscosity and isotope effects that identified the catalytic steps governing the steady-state rate of aminoglycoside inactivation. Together with the structural details of AAC{6')-li in complex with AcCoA, site-directed mutagenesis and related studies have also identified amino acid residues important to the chemistry of drug acetylation as well as those involved in the binding of aminoglycoside substrates, providing us with a better understanding of the molecular mechanism of AAC(6')-Ii catalysis. Our results to date have been applied to numerous inhibitor studies, which have resulted in the identification of cationic peptides and semisynthetic aminoglycosides that demonstrate potent inhibition of AAC(6')-Ii activity in vitro. Finally, the acetylation of E. faecium proteins by AAC(6')-Ii has been shown by both in vitro and in vivo analysis, complementing the structural and functional homology observed for this enzyme and protein acetyltransferases, in addition to supporting our hypothesis that AAC(6')-Ii may have an alternate function in the host bacteria. As a whole, these studies have extensively characterized the activity of AAC(6')-Ii, which is relevant to ongoing and future inhibitor studies and to an understanding of the similarities and differences among enzymes in the GCNS-related N-acetyltransferase superfamily.

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