|M.Sc Student||Tall Asaf|
|Subject||Biochemical Characterization of Aminoglycoside Modifying|
Enzyme APH(2")-AAC(6') with New Synthetic
Derivatives of Neomycin B
|Department||Department of Biotechnology||Supervisor||Professor Timor Baasov|
|Full Thesis text - in Hebrew|
One of the most important challenges facing modern medical science is the discovery of new antibiotics that are capable of confronting pathogenic bacteria having resistance mechanisms to a wide range of antibiotics. Aminoglycosides are highly potent, broad-spectrum antibiotics that exert their antibacterial effect by binding to the decoding aminoacyl site (A-site) of the bacterial 16S rRNA, and interfering with translational fidelity during protein synthesis. The major mechanism of aminoglycoside resistance is enzymatic modification of the amino or hydroxyl groups of these antibiotics. Modified aminoglycosides loose their ability to bind to the ribosome in high affinity and subsequently loose their antibacterial potential. The APH(2") - AAC(6') is a bifunctional enzyme forming part of the aminoglycoside modifying enzymes. This enzyme phosphorylates the 2"-hydroxyl of aminoglycosides, such as Kanamaycin A, as well as the 3'- and 3"'-hydroxyls of certain aminogltcosides, such as Neomycin B. In addition, the enzyme acetylates the 6'-amino group of the majority of these drugs. To tackle this problem, numerous derivatives of aminoglycosides, containing either monosaccharide or disaccharide at 5"-position were synthesized. A further number of derivatives, containing hydroxyl at 6'-position, were synthesized for the purpose of inhibiting acetylation at this position.
The goals of this research were an over expression, production and purification of the APH(2'')-AAC(6') enzyme, as well as determination of the kinetic parameters of this enzyme with the new derivatives. We used a plasmid DNA containing 6His-Tag at C-terminal or at N-terminal of the gene encoding APH(2") - AAC(6') enzyme. However, the recombinant enzyme obtained was lack of APH activity and displayed only AAC activity. Several comparative analyses of different plasmids with and without His-Tag confirmed that His-Tag attachment to the protein caused the loss of APH activity. The purified enzyme was used to evaluate AAC activity against a series of aminoglycoside drugs as well as their synthetic derivatives. Detailed kinetic study and determination of inhibition constants revealed several new synthetic analogs to be potent inhibitors of the enzyme.
This project is part of the long-term objective towards the development of new bifunctional aminoglycosides which in addition to targeting ribosomal RNA also resisting aminoglycoside-modifying enzymes. Understanding the molecular mechanisms that cause bacterial resistance, and subsequent design and examination of the mechanism-based inhibitors, is an important tool towards solving the problem of resistance and the development of new drugs.