The Catalytic and Conformational Cycle ofAquifex aeolicusKDO8P Synthase:  Role of the L7 Loop^,

Abstract

KDO8P synthase catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form the 8-carbon sugar KDO8P and inorganic phosphate (P i ). The X-ray structure of the wild-type enzyme shows that when both PEP and A5P bind, the active site becomes isolated from the environment due to a conformational change of the L7 loop. The structures of the R106G mutant, without substrates, and with PEP and PEP plus A5P bound, were determined and reveal that in R106G closure of the L7 loop is impaired. The structural perturbations originating from the loss of the Arg 106 side chain point to a role of the L2 loop in stabilizing the closed conformation of the L7 loop. Despite the increased exposure of the R106G active site, no abnormal reaction of PEP with water was observed, ruling out the hypothesis that the primary function of the L7 loop is to shield the active site from bulk solvent during the condensation reaction. However, the R106G enzyme displays several kinetic abnormalities on both the substrate side (smaller K m PEP , larger K i A5P and K m A5P ) and the product side (smaller K i Pi and K i KDO8P ) of the reaction. As a consequence, the mutant enzyme is less severely inhibited by A5P and more severely inhibited by P i and KDO8P. Simulations of the flux of KDO8P synthesis under metabolic steady-state conditions (constant concentration of reactants and products over time) suggest that in vivo R106G is expected to perform optimally in a narrower range of substrate and product concentrations than the wild-type enzyme.3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS, EC 2.5.1.55) catalyzes the irreversible condensation of phosphoenolpyruvate (PEP) 1 and arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate ( Figure 1) (1)(2)(3)(4)(5). This reaction is the first step in the biosynthesis of 3-deoxy-D-manno-octulosonate (KDO), an essential component of the lipopolysaccharide of all Gram-negative bacteria (6, 7). † This research was supported by U.S. Public Health Service Grants GM69840 to D.L.G. ‡ The structure-factor amplitudes and refined coordinates of substrate-free R106G, R106G with PEP, and PEP plus A5P, were deposited in the Protein Data Bank (entries 1T99, 1T96, 1T8X, respectively). 1 Abbreviations: KDO, 3-deoxy-D-manno-octulosonate; KDO8P, 3-deoxy-D-manno-octulosonate 8-phosphate; KDO8PS. 3-deoxy-Dmanno-octulosonate 8-phosphate synthase; PEP, phosphoenolpyruvate; A5P, arabinose 5-phosphate; 2-PGA, 2-phosphoglycerate; DSS, 2,2-dimethylsilapentane-5-sulfonic acid; DDM, difference-distance matrix; EDDM, error-scaled difference-distance matrix; SSR, sum of squared residuals; rmsd, root-mean-square deviation; H, helix; S, strand; L, loop. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThere are two types of KDO8PSs, distinguished by their requirement for divalent metals (Class II) or lack thereof (Class I) (8). In previous studies, we have determined the crystal structure of KDO8PS from the hyperthermophile Aquifex aeolicu...

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

“…KDO8PS was previously reported to follow an ordered sequential bi bi kinetic mechanism with PEP binding before A5P; ,, however, the metal ion’s place in the kinetic mechanism was not known. Because the metal ion affected inhibitor binding (see below), it was necessary to determine its place in the kinetic mechanism.…”

“…This agreed with previous studies showing that PEP binds before A5P, but which did not address metal binding. 34,49,50 E. coli DAHPS(Phe) follows the same mechanism. 15 A sequential ordered ter ter mechanism is likely to be universal among metal-dependent α-carboxyketose synthases.…”

Common basis for the mechanism of metallo and non-metallo KDO8P synthases

“…On the other hand, based on several structures of wild-type and mutant Aa. KDO8PS [14, 48, 49], we have proposed that in metallo KDO8PS the metal favors the deprotonation of a water molecule to form a hydroxide ion that attacks C2 PEP ; the ensuing carbanion at C3 PEP (C_ION, Fig. 1) would then facilitate attack onto C1 A5P .…”

New Targets for Antibacterial Design: Kdo Biosynthesis and LPS Machinery Transport to the Cell Surface

“…In its closed conformation, L7 loop interacts both with the A5P phosphate group through Ser197 and with the L2 loop. According to the proposed model, while the L7 loop acts as a lid controlling the access to the active site, the L2 loop behaves as the latch that blocks the L7 loop in position [126]. Obviously, it is expected that L7 loop plays the same role both in A. aeolicus and E. coli enzymes.…”