title: Fluorescence anisotropy-based measurement of Pseudomonas aeruginosa penicillin-binding protein 2 transpeptidase inhibitor acylation rate constants.
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.
authors.affiliation: Biology Department, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA 02451, USA. Electronic address: adam.shapiro@astrazeneca.com.
authors.forename: Adam B
authors.initials: AB
authors.lastname: Shapiro
authors.affiliation: Reagents and Assay Development, Discovery Sciences, AstraZeneca R&D Boston, Waltham, MA 02451, USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
authors.affiliation: Biology Department, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA 02451, USA.
authors.forename: Rong-Fang
authors.initials: RF
authors.lastname: Gu
authors.affiliation: Biology Department, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA 02451, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Thresher
mesh_terms: D000215:Acylation; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001896:Boron Compounds / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005454:Fluorescence Polarization; D007700:Kinetics; D046915:Penicillin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010406:Penicillins / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: C118961:BOCILLIN FL; D001426:Bacterial Proteins; D001896:Boron Compounds; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D011993:Recombinant Fusion Proteins
keywords: BOCILLIN; Fluorescence anisotropy; Fluorescence polarization; PBP2; Penicillin-binding protein; Transpeptidase