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Anal Biochem. 2014 Oct 15;463:15-22. doi: 10.1016/j.ab.2014.06.004. Epub 2014 Jun 16.

Fluorescence anisotropy-based measurement of Pseudomonas aeruginosa penicillin-binding protein 2 transpeptidase inhibitor acylation rate constants.

Author information

1
Biology Department, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA 02451, USA. Electronic address: adam.shapiro@astrazeneca.com.
2
Reagents and Assay Development, Discovery Sciences, AstraZeneca R&D Boston, Waltham, MA 02451, USA.
3
Biology Department, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA 02451, USA.

Abstract

High-molecular-weight penicillin-binding proteins (PBPs) are essential integral membrane proteins of the bacterial cytoplasmic membrane responsible for biosynthesis of peptidoglycan. They are the targets of antibacterial β-lactam drugs, including penicillins, cephalosporins, and carbapenems. β-Lactams covalently acylate the active sites of the PBP transpeptidase domains. Because β-lactams are time-dependent inhibitors, quantitative assessment of the inhibitory activity of these compounds ideally involves measurement of their second-order acylation rate constants. We previously described a fluorescence anisotropy-based assay to measure these rate constants for soluble constructs of PBP3 (Anal. Biochem. 439 (2013) 37-43). Here we report the expression and purification of a soluble construct of Pseudomonas aeruginosa PBP2 as a fusion protein with NusA. This soluble PBP2 was used to measure second-order acylation rate constants with the fluorescence anisotropy assay. Measurements were obtained for mecillinam, which reacts specifically with PBP2, and for several carbapenems. The assay also revealed that PBP2 slowly hydrolyzed mecillinam and was used to measure the rate constant for this deacylation reaction.

KEYWORDS:

BOCILLIN; Fluorescence anisotropy; Fluorescence polarization; PBP2; Penicillin-binding protein; Transpeptidase

PMID:
24945954
DOI:
10.1016/j.ab.2014.06.004
[Indexed for MEDLINE]

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