|Author:||Fung, Yik Hong|
|Title:||Understanding the roles of Zn1 ion in New Delhi metallo-beta-lactamase 1 by kinetic assays and molecular dynamics simulations of protein mutants|
|Subject:||Hong Kong Polytechnic University -- Dissertations|
Beta lactam antibiotics
|Pages:||xxi, 173 pages : color illustrations|
|Abstract:||New Delhi metallo-beta-lactamase 1 (NDM-1) was first detected in 2008 in India. NDM-1 is an enzyme that can hydrolyze a broad range of beta-lactam antibiotics, including the carbapenem family which is known as the antibiotics of last resort. As infections involving NDM-1 have great resistance to antibiotics, they are usually very difficult to treat. Therefore, bacteria carrying NDM-1 are referred as "super bugs". NDM-1 uses six amino-acid residues to bind two zinc ions in its active-site: H120, H122, H189 (Zn1 site) and D124, C208, H250 (Zn2 site). Multiple studies had shown that the Zn2 ion and the residues in the Zn2 site, such as D124 and C208, are very important in the hydrolysis reaction. On the other hand, the role of the Zn1 ion in NDM-1 is less studied. In this project, the roles of Zn1 ion were explored and its functions in NDM-1 were determined. Residue H120 of NDM-1 was mutated to alanine (A), asparagine (N) and glutamine (Q) in order to knock out the binding of the Zn1 ion. Interestingly, the Zn1 ion was found to remain in the enzyme after the mutations. The mutants were tested for their hydrolytic activities to six different beta-lactam antibiotics: ampicillin, penicillin G, cefotaxime, ceftazidime, imipenem and meropenem. The activities of the mutants were reduced from 6 to 810 folds for different antibiotics. Molecular dynamics simulations suggested that the Zn1 ion moved away from its original position due to mutations. This consequently moved the hydroxide ion and Zn2 ion away from the beta-lactam ring of the antibiotics and reduced the hydrolytic activities. For the H120A and H120N mutants, the bridging hydroxide ion moved 1.3 A away from its original position. This movement broke its hydrogen bond with the D124 residue and resulted in an unfavorable conformation for nucleophilic attack. For the H120Q mutant, the Zn2 ion was moved 2.13 A from its original location and this resulting position was not suitable for stabilizing the hydrolysis intermediate. My studies suggested that the exact location of the Zn1 ion is critical to the activity of NDM-1. The relative location of the Zn1 ion is important for the formation of the Zn1-OH-Zn2 bridge during hydrolysis. A specific location of the Zn1 ion is required to hold the hydroxide ion and the Zn2 ion in the correct positions to perform nucleophilic attack for the hydrolysis reaction and to stabilize the negatively charged intermediate.|
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