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Biochemistry, Biophysics, and Structural Biology Commons

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Chemistry Faculty Publications

Biochemistry

2012

Articles 1 - 4 of 4

Full-Text Articles in Biochemistry, Biophysics, and Structural Biology

Exploring The Role Of A Conserved Class A Residue In The Ω-Loop Of Kpc-2 Β-Lactamase: A Mechanism For Ceftazidime Hydrolysis Sep 2012

Exploring The Role Of A Conserved Class A Residue In The Ω-Loop Of Kpc-2 Β-Lactamase: A Mechanism For Ceftazidime Hydrolysis

Chemistry Faculty Publications

Gram-negative bacteria harboring KPC-2, a class A β-lactamase, are resistant to all β-lactam antibiotics and pose a major public health threat. Arg-164 is a conserved residue in all class A β-lactamases and is located in the solvent-exposed Ω-loop of KPC-2. To probe the role of this amino acid in KPC-2, we performed site-saturation mutagenesis. When compared with wild type, 11 of 19 variants at position Arg-164 in KPC-2 conferred increased resistance to the oxyimino-cephalosporin, ceftazidime (minimum inhibitory concentration; 32→128 mg/liter) when expressed in Escherichia coli. Using the R164S variant of KPC-2 as a representative β-lactamase for more detailed ...


Understanding The Molecular Determinants Of Substrate And Inhibitor Specificities In The Carbapenemase Kpc-2: Exploring The Roles Of Arg220 And Glu276 Aug 2012

Understanding The Molecular Determinants Of Substrate And Inhibitor Specificities In The Carbapenemase Kpc-2: Exploring The Roles Of Arg220 And Glu276

Chemistry Faculty Publications

β-Lactamases are important antibiotic resistance determinants expressed by bacteria. By studying the mechanistic properties of β-lactamases, we can identify opportunities to circumvent resistance through the design of novel inhibitors. Comparative amino acid sequence analysis of class A β-lactamases reveals that many enzymes possess a localized positively charged residue (e.g., R220, R244, or R276) that is critical for interactions with β-lactams and β-lactamase inhibitors. To better understand the contribution of these residues to the catalytic process, we explored the roles of R220 and E276 in KPC-2, a class A β-lactamase that inactivates carbapenems and β-lactamase inhibitors. Our study reveals that ...


Removal Of Uracil By Uracil Dna Glycosylase Limits Pemetrexed Cytotoxicity: Overriding The Limit With Methoxyamine To Inhibit Base Excision Repair, A. D. Bulgar, L. D. Weeks, Y. Miao, S. Yang, Yan Xu, C. Guo, S. Markowitz, N. Oleinick, S. L. Gerson, Lili Liu Jan 2012

Removal Of Uracil By Uracil Dna Glycosylase Limits Pemetrexed Cytotoxicity: Overriding The Limit With Methoxyamine To Inhibit Base Excision Repair, A. D. Bulgar, L. D. Weeks, Y. Miao, S. Yang, Yan Xu, C. Guo, S. Markowitz, N. Oleinick, S. L. Gerson, Lili Liu

Chemistry Faculty Publications

Uracil DNA glycosylase (UDG) specifically removes uracil bases from DNA, and its repair activity determines the sensitivity of the cell to anticancer agents that are capable of introducing uracil into DNA. In the present study, the participation of UDG in the response to pemetrexed-induced incorporation of uracil into DNA was studied using isogenic human tumor cell lines with or without UDG (UDG+/+/UDG−/−). UDG−/− cells were very sensitive to pemetrexed. Cell killing by pemetrexed was associated with genomic uracil accumulation, stalled DNA replication, and catastrophic DNA strand breaks. By contrast, UDG+/+ cells were >10 times more resistant to pemetrexed due ...


Development Of A ‘Clickable’ Non-Natural Nucleotide To Visualize The Replication Of Non-Instructional Dna Lesions, Edward A. Motea, Irene Lee, Anthony J. Berdis Jan 2012

Development Of A ‘Clickable’ Non-Natural Nucleotide To Visualize The Replication Of Non-Instructional Dna Lesions, Edward A. Motea, Irene Lee, Anthony J. Berdis

Chemistry Faculty Publications

The misreplication of damaged DNA is an important biological process that produces numerous adverse effects on human health. This report describes the synthesis and characterization of a non-natural nucleotide, designated 3-ethynyl-5-nitroindolyl-2′-deoxyriboside triphosphate (3-Eth-5-NITP), as a novel chemical reagent that can probe and quantify the misreplication of damaged DNA. We demonstrate that this non-natural nucleotide is efficiently inserted opposite an abasic site, a commonly formed and potentially mutagenic non-instructional DNA lesion. The strategic placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with an azide-containing fluorophore using ‘click’ chemistry. This reaction provides a facile way ...