Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/33424
Title: STRUCTURAL AND FUNCTIONAL STUDIES OF METHYLTRANSFERASES SGM AND NPMA THAT CONFER ANTIBIOTIC RESISTANCE AND THEIR INTERACTIONS WITH THE 30S RIBOSOMAL SUBUNIT
Authors: NILOFER HUSAIN
Keywords: Methyltransferase, Antibiotic Resistance, Ribosome, Crystallography, Mass Spectrometry, NpmA, Sgm
Issue Date: 20-Dec-2011
Source: NILOFER HUSAIN (2011-12-20). STRUCTURAL AND FUNCTIONAL STUDIES OF METHYLTRANSFERASES SGM AND NPMA THAT CONFER ANTIBIOTIC RESISTANCE AND THEIR INTERACTIONS WITH THE 30S RIBOSOMAL SUBUNIT. ScholarBank@NUS Repository.
Abstract: On a worldwide basis, infectious diseases are responsible for over one-third of all deaths. Aminoglycosides are bactericidal antibiotics that are widely used in treatment against severe infectious diseases caused by Gram-negative and Gram-positive bacteria. Aminoglycoside resistance is mainly acquired by pathogens by the expression of rRNA methyltransferases that can specifically methylate the bacterial ribosome at a particular antibiotic-binding site to prevent the binding of antibiotics. Sgm, from an antibiotic-producing bacterium and NpmA, identified in an Escherichia coli clinical isolate are methyltransferases that confer resistance to aminoglycosides (antibiotics). My PhD project mainly focused on Sgm (Arm family) and NpmA (Kam family) that specifically methylate the 16S rRNA of the small (30S) ribosomal subunit at G1405 and A1408, respectively. Sgm was crystallized and studied in complex with its cofactors S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). We then carried out structure-guided mutagenesis, isothermal titration calorimetry and protein¿RNA footprinting to develop a model of Sgm¿rRNA interactions and explain its mechanism of m7G1405 methylation in 16S rRNA (Husain N et al., (2010) Nucleic Acids Research 38, 4120-4132). This analysis can serve as a stepping stone towards developing drugs that would specifically block the activity of Arm methyltransferases and thereby re-sensitize pathogenic bacteria to aminoglycoside antibiotics. In addition, we have studied the structures of apo-NpmA and its complexes with SAM and SAH. We generated a number of NpmA mutants and studied their ability to bind the cofactor, to methylate A1408 in the 30S subunit, and to confer resistance to kanamycin in vivo. Residues D30, W107 and W197 were found to be essential. Moreover we analyzed the interactions between NpmA and the 30S subunit by footprinting experiments and computational docking (Husain N et al., (2011) Nucleic Acids Research 39, 1903-1918). NpmA is plasmid-encoded and can be transferred between pathogenic bacteria; therefore it poses a threat to the successful use of aminoglycosides in clinical practice. The results presented here will assist in the development of specific NpmA inhibitors that could restore the potential of aminoglycoside antibiotics. In continuation of our efforts to study the interaction of the methyltransferase with the 30S ribosomal subunit, we performed Hydrogen/Deuterium Mass Spectrometry (HDMS) experiment to map the region of the MTase that interacts with the ribosome. Our studies will lead to inactivate the antibiotic resistance methyltransferases and to retain the function of antibiotics.
URI: http://scholarbank.nus.edu.sg/handle/10635/33424
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