Contributions of Aspartate 49 and Phenylalanine 142 Residues of a Tight Binding Inhibitory Protein of β-Lactamases

Abstract

␤-Lactamases are bacterial enzymes that hydrolyze ␤-lactam antibiotics to render them inactive. The ␤-lactamase inhibitor protein (BLIP) of Streptomyces clavuligerus, is a potent inhibitor of several ␤-lactamases, including the TEM-1 enzyme (K i ‫؍‬ 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 (K i ) for both mutants were determined. Each mutation increases the K i for BLIP inhibition of TEM-1 ␤-lactamase approximately 100-fold. To address how these two positions effect the specificity of ␤-lactamase binding, K i values were determined for the interaction of wild-type BLIP, as well as the D49A and F142A mutants, with two extended spectrum ␤-lactamases (the G238S and the E104K TEM variants). Positions 104 and 238 are located in the BLIP/␤-lactamase interface. Interestingly, the three BLIP proteins inhibited the G238S ␤-lactamase mutant to the same degree that they inhibited TEM-1. However, wild-type BLIP has a higher K i for the E104K ␤-lactamase mutant, suggesting that interactions between BLIP and ␤-lactamase residue Glu-104 are important for wild-type levels of BLIP inhibition.

Functional Analysis of Active Site Residues of the Fosfomycin Resistance Enzyme FosA from Pseudomonas aeruginosa

“…Cloning of fosA-The fosA gene (408 bp) was PCR-amplified with Taq polymerase (Sigma) and the primers FosNde 5Ј-ACGATACATATGCT-TACCGGTCTCAAT-3Ј and FosXho 5Ј-ACGATACTCGAGCTAGTCG-GCGAAACG-3Ј (with added NdeI and XhoI restriction sites underlined) or FosSac 5Ј-ACGATAGAGCTCATGCTTACCGGTCTCAAT-3Ј and FosXba 5Ј-ACGATATCTAGACTAGTCGGCGAAACG-3Ј (with added SacI and XbaI sites underlined) from P. aeruginosa genomic DNA and ligated into the multiple cloning site of pET-24a(ϩ) (NdeI, XhoI) (Novagen) to yield plasmid pFOS2 or pTP123 (SacI, XbaI) (27) to yield plasmid pFOS1. The nucleotide sequence of pFOS1 and pFOS2 was confirmed to be the same as GenBank TM accession number NC002516.…”

Structural and Biochemical Evidence That a TEM-1 β-Lactamase N170G Active Site Mutant Acts via Substrate-assisted Catalysis

“…The sense and antisense strand primers outside of the bla TEM-1 gene were PD-bla1 and TEM-XbaI-bot, which contained the SacI and XbaI sites, respectively, at the 5Ј-ends as follows: TEM-N170X, top, 5Ј-GGG AAC CGG AGC TGN NNG AAG CCA TAC CAA A-3Ј; TEM-N170X, bottom, 5Ј-CGT TTG GTA TGG CTT CNN NCA GCT CCG GTT CC-3Ј; PD-bla1, 5Ј-CGG GGA GCT CGT TTC TTA GAC GTC AGG TGG CAC-3Ј; and TEM-XbaI, bottom, 5Ј-GCG TGG TGC AAG TCT AGA TTA CCA ATG CTT AA-3Ј. The pBG66 plasmid contains the wild-type bla TEM-1 gene and was used as template for the PCRs. The final PCR product was digested with SacI and XbaI restriction endonucleases and ligated into SacI-XbaI-cut pTP123 plasmid (32). The ligation mixtures were used to transform E. coli XL-1-Blue (Stratagene) by electroporation, and colonies were selected on LB agar plates containing 12.5 g/ml chloramphenicol.…”

“…The sense and antisense strand primers outside of the bla TEM-1 gene were PD-bla1 and TEM-XbaI-bot, which contained the SacI and XbaI sites, respectively, as described for the library construction above. The final PCR product was digested with SacI and XbaI and ligated into SacIXbaI cut pTP123 plasmid (32). The ligation mixtures were used to transform E. coli XL-1-Blue by electroporation, and colonies were selected on LB agar plates containing 12.5 g/ml chloramphenicol.…”

Identification of the β-Lactamase Inhibitor Protein-II (BLIP-II) Interface Residues Essential for Binding Affinity and Specificity for Class A β-Lactamases

“…In addition, contact residues have also been identified as specificity determinants if the substitution of the residue changes the binding affinity for one binding partner relative to another (8,9). The relationship between specificity and affinity is an active area of research and requires model systems, such as the human growth hormone-receptor, barnase-barstar, and BLIP-␤-lactamase, to provide insights (4,5,10,11).…”