Campylobacter jejuni KDO8P Synthase, Its Inhibition by KDO8P Oxime, and Control of the Residence Time of Slow-Binding Inhibition

• Published in: Biochemistry (Easton) 2018-09, Vol.57 (36), p.5327-5338
• Main Authors: Gama, Simanga R, Balachandran, Naresh, Berti, Paul J
• Format: Article
• Language: English
• Subjects: Aldehyde-Lyases - antagonists & inhibitors; Bacterial Proteins - antagonists & inhibitors; Binding Sites; Campylobacter jejuni - enzymology; Catalysis; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Kinetics; Models, Molecular; Oximes - chemistry; Oximes - pharmacology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.8b00748

3-Deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the first step of lipopolysaccharide biosynthesis, namely condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P), to produce KDO8P. We have characterized Campylobacter jejuni KDO8P synthase and its inhibition by KDO8P oxime. It was metal-dependent and homotetrameric and followed a rapid equilibrium sequential ordered ter ter kinetic mechanism in which Mn2+ bound first, followed by PEP and then A5P. It was inhibited by KDO8P oxime, an analogue of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) oxime, a transition-state mimic of DAHP synthase. Inhibition was uncompetitive-like with respect to Mn2+ and competitive with respect to PEP and A5P. It displayed both fast-binding inhibition (K i = 10 μM) and slow-binding inhibition (K i* = 0.57 μM). The residence times on the enzyme (t R) ranged from 27 min in the absence of free inhibitor to 69 h with excess inhibitor. The dependence of t R on the free inhibitor concentration suggested intersubunit communication within the homotetramer between high- and low-affinity sites. This confirms the generality of the oxime functional group, a small, neutral phosphate bioisostere, as an α-carboxyketose synthase inhibitor and highlights the challenge that intersubunit communication poses to effective inhibition.