INVESTIGATION OF THE N-TERMINUS OF RESISTANCE ENZYME AAC(6')-IB

Amikacin, a semisynthetic aminoglycoside antibiotic, is used to treat bacterial infections caused by antibiotic resistant bacteria. However, AAC(6’)-Ib (aminoglycoside 6’N- acetyltransferase) is an AME (aminoglycoside modifying enzyme) found bacteria that acetylates amikacin, causing inactivation of the aminoglycoside and preventing cell death. There are numerous variants of AAC(6’)-Ib that can be found in nature and between these variants is a high amount of amino acid variation at the N-terminus of the enzymes. The size of N-terminus amino acid variations has been linked to small differences in relative acetylation efficiency levels of the AAC(6’)-Ib variants but it is unknown if the N-terminus amino acids are important to the enzymes. This project investigated the N-terminus of AAC(6’)-Ib to determine if it had a purpose and explored what that purpose could be. E. coli strains expressing N-terminus deletion mutant AAC(6’)-Ib enzymes were created. The presence of the deletion mutant enzymes and their inactivation activity against amikacin were measured using western blot analysis and enzymatic activity assays respectively. It was found that deletion mutant AAC(6’)-Ib enzymes lacking twenty-five or more N-terminus amino acids were not detected in the host cells. Additional examination showed the lack of presence of the enzymes was not due to loss of solubility nor decreased enzyme stability. The purpose of the N-terminus of AAC(6’)-Ib enzymes relates to the presence of the enzymes in bacteria, likely increasing enzyme expression.