The emergence and dissemination of carbapenem-resistant Gram-negative pathogens is a significant source of healthcare-associated morbidity and mortality. Carbapenemases (i.e., carbapenem-hydrolyzing -lactamases) that potentially inactivate all -lactams severely limit the antimicrobials available to treat infections with these bacteria. Presently, two major carbapenemases are emerging in Klebsiella and are a major threat to our antibiotic armamentarium: the Klebsiella pneumoniae carbapenemase (KPC-2), and the New Delhi Metallo--lactamase (NDM-1 MBL). KPC is a class A carbapenemase that hydrolyzes imipenem, ertapenem, meropenem, doripenem, and - lactamase inhibitors and is found in almost every major tertiary care facility in the US. In order to design future -lactams to combat these imminent threats, we need to understand how the substrate profile and susceptibility patterns of strains that possess KPC and NDM-1 will change as a result of substitutions in hot spots in the -lactamase (i.e., &-loop, amino acid residues 164 to 179 in KPC-2, and the loop regions and entrance to the active site of NDM-1). These challenges frame the research direction that will be addressed herein. The major research questions we will ask in this Merit Proposal are: 1) If substitutions in the &-loop arise in KPC-2, will they pose a significant clincal threat by representing novel phenotypes that will present even greater challenges to the clinician? 2) How do unique structural features of NDM-1 MBL impact the ability of the -lactamase to hydrolyze -lactams? To answer the first question our plan is to determine the effect of substitutions in the &-loop at amino acid positions R164, E166, N170, and D179 in KPC-2 and ascertain how these mutations alter microbiological phenotype, protein stability and catalytic mechanism. To address the second question we will use mutagenesis to remove, in a stepwise manner, the distinctive additional sequence at amino acid positions 163 to 166 ( 163-166) in NDM-1 and assess the impact of these deletions on resistance (phenotype), hydrolysis of standard -lactam and carbapenem substrates, and protein stability. Next, we will use mutagenesis to replace (site-saturation) and to delete F70 ( 70) in NDM-1, an amino acid at the entrance of the active site and assess the activity of this variant and its role in protein stability and catalysis. By focusing on the carbapenemases of K. pneumoniae we will provide the critical knowledge that leads to novel therapies. Our laboratory is actively defining the amino acid sequence requirements of KPC-2 and this new project will stand out as an important undertaking in the further exploration of the role of the &-loop and in the biochemical study of NDM-1. Achieving our objectives will advance the general understanding needed for the medicinal chemist to design effective therapeutic agents that will act as substrates and inhibitors against these versatile -lactamases and help us devise antibiotic treatment regimens that will assist physicians faced with this serious and emerging infectious disease threats.