Please use this identifier to cite or link to this item:
http://hdl.handle.net/11375/22815
Title: | CHARACTERIZATION OF THE METAL-DEPENDENT KDO8P SYNTHASE FROM CAMPYLOBACTER JEJUNI AND INHIBITION BY KDO8P OXIME, A NOVEL SLOW-BINDING INHIBITOR |
Other Titles: | CAMPYLOBACTER JEJUNI KDO8PS: A METAL-DEPENDENT KDO8PS |
Authors: | Gama, Simanga R. |
Advisor: | Berti, Paul J. |
Department: | Chemistry and Chemical Biology |
Keywords: | Campylobacter jejuni KDO8PS Kinetics;KDO8P oxime inhibitor, novel slow-binding inhibitor, residence time |
Publication Date: | Nov-2017 |
Abstract: | Antibiotic resistance is a worldwide threat to human health yet fewer new antibiotics are being approved. New antimicrobial drugs are urgently required. 3 Deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS) is a target for antimicrobial drug design. KDO8PS catalyzes the condensation of D-arabinose-5 phosphate (A5P) with phosphoenolpyruvate (PEP) to produce KDO8P. KDO8PS catalyzes the first committed step in the lipopolysaccharides (LPS) biosynthesis pathway in Gram-negative bacteria and is critical for bacterial pathogenicity/virulence. We have characterized KDO8PS from Campylobacter jejuni (cjKDO8PS), a new metal-dependent KDO8P synthase (KDO8PS). cjKDO8PS is a tetramer in solution and optimally active at pH 7.5 and 60 °C. We have kinetically established that cjKDO8PS follows a rapid equilibrium sequential ordered ter ter kinetic mechanism, where Mn2+ binds first, followed by PEP, then A5P. Pi dissociates first, before KDO8P, then Mn2+. cjKDO8PS was inhibited by KDO8P oxime, a novel slow tight-binding inhibitor. KDO8P oxime is a competitive inhibitor with respect to PEP and A5P, but uncompetitive with respect to Mn2+, with Ki = 10 ± 1 μM and an ultimate Ki* = 0.28 ± 0.10 μM. KDO8P oxime has a residence time (tR) of 5 days on the enzyme, a parameter that is highly correlated to in vivo efficacy. Crystallization conditions for the cjKDO8PS‧Mn2+‧KDO8P oxime complex have been found and can be optimized to obtain a crystal structure that shows how KDO8P oxime interacts with the active sites. |
URI: | http://hdl.handle.net/11375/22815 |
Appears in Collections: | Open Access Dissertations and Theses |
Files in This Item:
File | Description | Size | Format | |
---|---|---|---|---|
Gama_Simanga_R_2017November_PhD.pdf | 5.17 MB | Adobe PDF | View/Open |
Items in MacSphere are protected by copyright, with all rights reserved, unless otherwise indicated.