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

Abstract

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 Mn 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 Mn and competitive with respect to PEP and A5P. It displayed both fast-binding inhibition ( K = 10 μM) and slow-binding inhibition ( K* = 0.57 μM). The residence times on the enzyme ( t) ranged from 27 min in the absence of free inhibitor to 69 h with excess inhibitor. The dependence of t 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.

Network-Based Metabolism-Centered Screening of Potential Drug Targets in Klebsiella pneumoniae at Genome Scale

“…More recently, KDO8P oxime was evaluated in terms of the inhibitory capacity, which supported a greater understanding of the binding kinetics. This may lead to more efficient KdsA inhibitors (Gama et al, 2018). Identification of the KdsA protein at the top of the ranked putative target list in the current study and studies on KdsA inhibitors encouraged us to investigate K. pneumoniae KdsA inhibitors.…”

NeuNAc Oxime: A Slow-Binding and Effectively Irreversible Inhibitor of the Sialic Acid Synthase NeuB

“…Initial velocities were measured by following inorganic phosphate (P i ) production with the Malachite green/ammonium molybdate colorimetric assay. , Rate assays were typically conducted with 150 nM NeuB in reaction buffer [50 mM Tris-acetate (pH 8.3), 100 μM tris­(2-carboxyethyl)­phosphine (TCEP), and 0.1 mg/mL bovine serum albumin] at 37 °C. When measuring the Michaelis–Menten kinetic parameters, two substrate concentrations were fixed while the third was varied.…”

“…It catalyzes the metal ion-dependent condensation of N -acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP), passing through a tetrahedral intermediate (THI) to form NeuNAc (Figure ). It is the archetypal member of the NeuB superfamily of α-carboxyketose synthases, which includes 2-keto-3-deoxy- d - arabino -heptulosonate-7-phosphate (DAHP) and 2-keto-3-deoxy- d - manno -octulosonate-8-phosphate (KDO8P) synthases, − which are also antimicrobial targets. DAHP synthase ,, and KDO8P synthase , follow sequential ordered ter ter kinetic mechanisms, which is likely universal throughout the NeuB superfamily.…”

“… It is the archetypal member of the NeuB superfamily of α-carboxyketose synthases, which includes 2-keto-3-deoxy- d - arabino -heptulosonate-7-phosphate (DAHP) and 2-keto-3-deoxy- d - manno -octulosonate-8-phosphate (KDO8P) synthases, − which are also antimicrobial targets. DAHP synthase ,, and KDO8P synthase , follow sequential ordered ter ter kinetic mechanisms, which is likely universal throughout the NeuB superfamily. NeuB is active as a homodimer, with a domain-swapped structure composed of an N-terminal TIM barrel catalytic domain and a C-terminal domain that is homologous to a type III antifreeze protein (AFP) and contributes its R314 side chain to the neighboring subunit’s active site. , …”

“…α-Carboxyketose synthases have been the targets of inhibition since the 1970s; however, highly potent inhibitors remain rare, and the only potent, mechanism-based inhibitor of NeuB characterized to date is a slow-binding THI mimic ( 1 ) with a K i * of 3 μM (Figure , bottom) . We recently demonstrated that the DAHP oxime is a transition state mimic and potent inhibitor of DAHP synthase, with a K i of 1.5 μM, and a residence time in the active site, t R , of 83 min, , while KDO8P oxime inhibits KDO8P synthase with a K i * down to 0.57 μM and residence times of up to 69 h . Long residence times are an increasingly important inhibitor design criterion due to the correlation between slow dissociation and drug efficacy in vivo. − The oxime functional group of DAHP oxime, plus two crystallographic waters, structurally mimics the THI’s phosphate group and functionally mimics the transition state for THI breakdown …”