Contributions of aspartate 49 and phenylalanine 142 residues of a tight binding inhibitory protein of beta-lactamases.
beta-Lactamases are bacterial enzymes that hydrolyze beta-lactam antibiotics to render them inactive. The beta-lactamase inhibitor protein (BLIP) of Streptomyces clavuligerus, is a potent inhibitor of several beta-lactamases, including the TEM-1 enzyme (Ki = 0.6 nM). Evidence from the TEM-1/BLIP co-crystal suggests that two BLIP residues, Asp-49 and Phe-142, mimic interactions made by penicillin G when bound in the active site of TEM-1. To determine the importance of these two residues, a heterologous expression system for BLIP was established in Escherichia coli. Site-directed mutagenesis was used to change Asp-49 and Phe-142 to alanine, and inhibition constants (Ki) for both mutants were determined. Each mutation increases the Ki for BLIP inhibition of TEM-1 beta-lactamase approximately 100-fold. To address how these two positions effect the specificity of beta-lactamase binding, Ki values were determined for the interaction of wild-type BLIP, as well as the D49A and F142A mutants, with two extended spectrum beta-lactamases (the G238S and the E104K TEM variants). Positions 104 and 238 are located in the BLIP/beta-lactamase interface. Interestingly, the three BLIP proteins inhibited the G238S beta-lactamase mutant to the same degree that they inhibited TEM-1. However, wild-type BLIP has a higher Ki for the E104K beta-lactamase mutant, suggesting that interactions between BLIP and beta-lactamase residue Glu-104 are important for wild-type levels of BLIP inhibition.
Petrosino
J
J
Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA.
Rudgers
G
G
Gilbert
H
H
Palzkill
T
T
D001224:Aspartic Acid / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D016296:Mutagenesis; D010649:Phenylalanine / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D013302:Streptomyces / Q000378:metabolism; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry
D001426:Bacterial Proteins; D017931:DNA Primers; D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; C065596:beta-lactamase-inhibitor protein, Streptomyces; D001224:Aspartic Acid; D010649:Phenylalanine; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1