Specific interaction between beta-lactams and soluble penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus: development of a chromogenic assay

Roychoudhury, Siddhartha; Kaiser, R. E.; Brems, David N.; Yeh, Wu-Kuang

doi:10.1128/aac.40.9.2075

Cited by 15 publications

(4 citation statements)

References 14 publications

(20 reference statements)

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“…Some previous studies of this enzyme have also suggested that it becomes more susceptible to precipitation after acylation by β-lactams. For example, Roychoudhury et al [15] have routinely observed the precipitation of sPBP2a at concentrations above 1 µM by a variety of β-lactams in 0.05 M phosphate buffer including 0.1 M KCl at pH 8.0 and 37 mC. Indeed, they have described a chromogenic assay that relies on this fact.…”

Section: Preparation and Properties Of Spbp2amentioning

confidence: 99%

“…We performed initial kinetic studies on sPBP2a employing nitrocefin because of the large change in absorption spectrum on the opening of its β-lactam ring [13]. Roychoudhury et al [15] have also employed nitrocefin to produce a chromophoric precipitate for their assay. First, we explored conditions under which sPBP2a acylation could be monitored in homogeneous solution without protein precipitation.…”

Section: Preparation and Properties Of Spbp2amentioning

confidence: 99%

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Reaction of soluble penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus with β-lactams and acyclic substrates: kinetics in homogeneous solution

Graves-Woodward

Pratt

1998

Biochemical Journal

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The kinetics of reaction of solubilized penicillin-binding protein 2a (sPBP2a) of methicillin-resistant Staphylococcus aureus with a variety of beta-lactams and acyclic species was studied in homogeneous aqueous solution at 37 degreesC in 25 mM Hepes buffer, pH7.0, containing 1 M NaCl. Under these conditions, but not at lower salt concentrations, protein precipitation did not occur either during or after the reaction. The reactions of beta-lactams in general could be monitored by competition with a chromophoric beta-lactam, nitrocefin, or directly in certain cases by protein fluorescence. Rate constants for reaction of a wide variety of beta-lactams are reported. The interactions are characterized by a slow second-order acylation reaction followed by a slower deacylation. For example, the rate constants for benzylpenicillin were 12 M-1.s-1 and 3x10(-5) s-1 respectively. The acylation is slow in comparison with those of normal non-resistant high-molecular-mass penicillin-binding proteins. sPBP2a also seemed to catalyse the slow hydrolysis of a variety of acyclic depsipeptides but not that of a d-Ala-d-Ala peptide. The reactions with certain depsipeptides also led to protein precipitation. These reactions were, however, not affected by prior blockage of the beta-lactam-binding site by benzylpenicillin and thus might take place elsewhere on the enzyme. Two classes of potential transition- state analogue inhibitors, phosphonate monoesters and boronates, seemed to have little effect on the rate of reaction of sPBP2a with nitrocefin and therefore seem to have little affinity for the beta-lactam-binding/D,D-peptidase site.

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Section: Preparation and Properties Of Spbp2amentioning

confidence: 99%

Section: Preparation and Properties Of Spbp2amentioning

confidence: 99%

Reaction of soluble penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus with β-lactams and acyclic substrates: kinetics in homogeneous solution

Graves-Woodward

Pratt

1998

Biochemical Journal

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“…The catalytic activity of PBP2a can be monitored at 482 nm using nitrocefin, a colorimetric substrate (24) . 10 μM BLIP-II was incubated with 2 μM PBP2a in 25 mM HEPES, 1.0 M NaCl for 45 minutes at room temperature before nitrocefin was added.…”

Section: Methodsmentioning

confidence: 99%

BLIP-II Employs Differential Hotspot Residues To Bind Structurally Similar Staphylococcus aureus PBP2a and Class A β-Lactamases

Adamski

Palzkill

2017

Biochemistry

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The interaction of β-lactamase inhibitory protein II (BLIP-II) with β-lactamases serves as a model system to investigate the principles underlying protein-protein interactions. Previous studies have focused on identifying the determinants of binding affinity and specificity between BLIP-II and class A β-lactamases. However, interactions between BLIP-II and other bacterial proteins have yet to be explored. Here, we provide evidence that BLIP-II binds penicillin binding protein 2a (PBP2a) from methicillin resistant Staphylococcus aureus (MRSA) with a KD in the low micromolar range. In comparison to the binding constants for the potent interaction between BLIP-II and TEM-1 β-lactamase (KD= 0.5 pM), the on-rate for BLIP-II binding PBP2a is 44,000 times slower and the off-rate is 170 times faster. Therefore, a slow association rate is a limiting factor for the potency of the interaction between BLIP-II and PBP2a. Results from alanine scanning mutagenesis of the predicted interface residues of BLIP-II indicate that charged residues on the periphery of the BLIP-II interface play a critical role for binding PBP2a in contrast to previous findings that aromatic residues at the center of the BLIP-II interface are critical for the interaction with β-lactamases. Interestingly, many of the alanine mutants at the BLIP-II interface increase kon for binding PBP2a, consistent with the association rate being a limiting factor for affinity. In summary, the results of the study reveal that BLIP-II binds PBP2a, although weakly compared to binding of β-lactamases, and provides insights into the different binding strategies used for these targets.

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“…The most commonly used is a screen for agents that compete with a labeled β-lactam for binding to the PBP 21,22 or cleavage of a chromogenic β-lactam on binding to the PBP. 23 However, these assays are not truly reflective of the enzyme activity, and in the light of widespread resistance to the β-lactams, a screen that detects new agents of the same class is of little value.…”

Section: Introductionmentioning

confidence: 99%

Microplate Assay for Inhibitors of the Transpeptidase Activity of PBP1b of Escherichia coli

Jha

Sousa

2006

SLAS Discovery

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The transpeptidase (TP) activity of penicillin-binding proteins (PBPs), target of the β-lactam antibiotics, is a well-validated antibacterial drug target. The TP activity of PBP1b converts un-cross-linked peptidoglycan to the cross-linked form. Directly measuring TP activity is difficult because cross-linked and un-cross-linked peptidoglycan have very similar chromatographic properties. The authors report a microdilution plate method to directly measure the TP enzyme activity, uncoupled from the transglycosylase (TG), for detection of TP inhibitors. Escherichia coli membranes were incubated with 100 mM ampicillin, followed by removal of unbound ampicillin. The substrate for the TP, un-cross-linked peptidoglycan, was prepared by incubating these membranes with peptidoglycan sugar precursors, 1 of which was radiolabeled. Subsequently, solubilized PBP1b was added and TP activity assayed. The cross-linked peptidoglycan formed was monitored by addition of wheat germ agglutinin scintillation proximity assay beads plus N-laurylsarcosine, which selectively captures cross-linked peptidoglycan. The PBP1b-catalyzed activity was inhibited by penicillin G but not by cephalexin or cephradine, which have higher affinity for PBP1a. Moenomycin, a TG inhibitor, also inhibited TP activity. Because this is a true enzyme assay, it has the potential to detect novel, non-β-lactam TP inhibitors and could lead to the discovery of new antibacterial agents. (Journal of Biomolecular Screening

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Specific interaction between beta-lactams and soluble penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus: development of a chromogenic assay

Cited by 15 publications

References 14 publications

Reaction of soluble penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus with β-lactams and acyclic substrates: kinetics in homogeneous solution

Reaction of soluble penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus with β-lactams and acyclic substrates: kinetics in homogeneous solution

BLIP-II Employs Differential Hotspot Residues To Bind Structurally Similar Staphylococcus aureus PBP2a and Class A β-Lactamases

Microplate Assay for Inhibitors of the Transpeptidase Activity of PBP1b of Escherichia coli

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