TAILIEUCHUNG - Báo cáo khóa học: Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin-binding protein 4

The penicillin-binding proteins (PBPs) are ubiquitous bac-terial enzymes involved in cell wall biosynthesis,and are the targets of theb-lactam antibiotics. The low molecular mass Neisseria gonorrhoeaePBP 4 (NG PBP 4) is the fourthPBP revealed in the gonococcal genome. NG PBP 4 was cloned, overexpressed,purified,and characterized for b-lactam binding,DD-carboxypeptidase activity,acyl-donor substrate specificity,transpeptidase activity, inhibitionby a number of active site directed reagents,and pHprofile | Eur. J. Biochem. 271 -3 2 2004 FEBS 2003 doi Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin-binding protein 4 Miglena E. Stefanova1 Joshua Tomberg2 Christopher Davies3 Robert A. Nicholas2 and William G. Gutheil1 division of Pharmaceutical Sciences University of Missouri-Kansas City USA department of Pharmacology University of North Carolina at Chapel Hill North Carolina USA 3Department of Biochemistry Medical University of South Carolina Charleston South Carolina USA The penicillin-binding proteins PBPs are ubiquitous bacterial enzymes involved in cell wall biosynthesis and are hie targets of the b-lactam antibiotics. The low molecular mass Neisseria gonorrhoeae PBP 4 NG PBP 4 is the fourth PBP revealed in the gonococcal genome. NG PBP 4 was cloned overexpressed uurified anh clictacleri ed for b-lactam binding DD-carboxypeptidase activity acybdonor subst rate specificity rranspepiid ase activity inhibition by a number of active site directed reagents and pH prolife. NG PBP 4 was efficiently acylated by penicillin 30 000 M-1-s-1 . Against a setoffive a-and e-substituted L-Lys-D-Ala-D-Ala substrates NG PBP 4 exhibited wide variation in specificity with a preference for Ne-acylated substrates suggesting a possible preference for crosslinked pentapeptide substrates in the cell wall. Substrates with an Ne-Cbz group demonstrated pronounced substrate inhibition. NG PBP 4 showed 30-fold higher activity against the depsipeptide Lac-ester substrate than against the analogous peptide substrate an indication that k2 acylation is rate determining for carboxypeptidase activity. No transpeptidase activity was apparent in a model transpeptidase reaction. Among a number of active site-directed agents N-chlorosuccinimide elastinal iodoaceta-mide iodoacetic acid and phenylglyoxal gave substantial inhibition and methyl boronic acid gave modest inhibition. The pH profile for activity against Ac2-L-Lys-D-Ala-D-Ala kcat

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