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

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 ( Ki = 10 μM) and slow-binding inhibition ( Ki* = 0.57 μM). The residence times on the enzyme ( tR) ranged from 27 min in the absence of free inhibitor to 69 h with excess inhibitor. The dependence of tR 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.

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