Campylobacter jejuni KDO8P Synthase, Its Inhibition by KDO8P

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Article Cite This: Biochemistry XXXX, XXX, XXX−XXX

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Campylobacter jejuni KDO8P Synthase, Its Inhibition by KDO8P Oxime, and Control of the Residence Time of Slow-Binding Inhibition Simanga R. Gama,† Naresh Balachandran,† and Paul J. Berti*,†,‡ †

Department of Chemistry & Chemical Biology and ‡Department of Biochemistry & Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada

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S Supporting Information *

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 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.

and waters in the active site was lower than for the dianionic phosphate monoester group. Thus, DAHP oxime mimicked a phosphate group both structurally and functionally. Slow dissociation, a necessary corollary of slow-binding inhibition, is increasingly associated with drugs’ in vivo efficacy.17−20 There is a correlation between inhibitors’ residence time on the enzyme (tR = 1/koff) and in vivo efficacy, as it permits the enzyme to remain inhibited even after the bulk of the inhibitor has been metabolized or excreted. We report that Campylobacter jejuni KDO8PS, a Class II KDO8PS,21 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, exhibiting both fast-binding inhibition, with a Ki of 10 μM, and slow-binding inhibition with residence times of up to 69 h (2.9 days) and an ultimate inhibition constant, Ki*, of 570 nM. This is the longest residence time to date for a KDO8PS inhibitor. The cooperativity of the concentration dependence of slow-binding inhibition and the dependence of tR on the free inhibitor

3-Deoxy- D -manno-2-octulosonate-8-phosphate synthase (KDO8PS) catalyzes the first committed step in lipopolysaccharide biosynthesis in Gram-negative bacteria1−3 and is an antimicrobial target.4−8 It catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) and arabinose 5-phosphate (A5P) that passes through a tetrahedral intermediate (THI) to produce KDO8P and inorganic phosphate (Pi) (Scheme 1). It is a member of the NeuB superfamily of α-carboxyketose synthases, which includes 3-deoxy-D-arabino-heptulosonate-7phosphate synthase (DAHPS)9 and NeuB, a sialic acid synthase.10 Inhibitor design for α-carboxyketose synthases has tended to focus on mimicking the THI 11−13 or the presumed oxacarbenium ion-like transition state for THI formation11,14 and has achieved Ki values down to the high nanomolar range. Recently, DAHP oxime was reported to be a slow, tightbinding DAHPS inhibitor.15 In the crystal structure, the oxime functional group, combined with two crystallographic waters, structurally mimicked the phosphate group of the THI/PEP. Linear free energy relationship analysis showed a correlation between the specificity constant, kcat/(KM,MnKM,PEPKM,E4P), and Ki, meaning that DAHP oxime mimics one of the transition states, presumably that for departure of Pi from the THI.16 The slope of the correlation was Cu2+ (Figure 2). Cu2+ was weakly activating at 200 μM but had no activity at 1 mM. This pattern of metal ion preference was typical of metaldependent KDO8PSs.21,42−48 Kinetic Mechanism. KDO8PS was previously reported to follow an ordered sequential bi bi kinetic mechanism with PEP binding before A5P;34,49,50 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. KDO8PSwt’s kinetic mechanism was analyzed essentially as described previously for E. coli DAHPS(Phe).15,26 The metal ion can be treated as a substrate in initial velocity measurements even though, strictly, it is an essential activator. Initial velocities were first fitted using a random order of metal binding and the steady-state approximation, using E

DOI: 10.1021/acs.biochem.8b00748 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry Table 1. KDO8PS Kinetic Constants parametera kcat(re) KM,Mn(re) KM,PEP(re) KM,A5P(re) kcat/(KM,MnKM,PEPKM,A5P) kcat(1s)b KM,Mn(1s) KM,PEP(1s) KM,A5P(1s)

KDO8PSwt Rapid Equilibrium Sequential Ordered Ter Ter 2.4 ± 0.1 s−1 130 ± 30 μM 650 ± 140 μM 21 ± 4 μM (1.4 ± 0.2) × 1012 M−3 s−1 Single Substrate 2.1 ± 0.3 s−1 16 ± 3 μM 48 ± 7 μM 36 ± 6 μM

KDO8PSH6 1.2 ± 0.1 s−1 6.4 ± 1.5 μM 900 ± 80 μM 14 ± 1 μM (1.4 ± 0.1) × 1013 M−3 s−1 1.2 ± 0.1 s−1 2.8 ± 0.5 μM 86 ± 16 μM 19 ± 6 μM

a

Legend: re, rapid equilibrium (eq 1); 1s, single substrate (eq 2); ss, steady state (see Table S4). bAverage of independent kcat values for each substrate.

literature KM values were significantly lower than those reported here for the full mechanism but similar to the single-substrate KM values (Table 1).4,33,43,44,51 This could be due to different sources or reaction conditions; however, literature KM values frequently only cite the “Michaelis− Menten” equation, suggesting that the single-substrate equation was used. Substrate Binding by ITC. The order and competitiveness of substrate and inhibitor binding were examined using ITC titrations with KDO8PSH6 (Figure 4). Given some ligands’ high Kd values and the limits on KDO8PSH6’s solubility, the experimental c values (ratio of analyte concentration to Kd) were in the range of 0.1−9, well below the optimal value of >40.30 At low c values, Kd and especially n (stoichiometry) become poorly defined. At c values of 300 μM, indicating competition between KDO8P oxime and PEP G

DOI: 10.1021/acs.biochem.8b00748 Biochemistry XXXX, XXX, XXX−XXX

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the preformed E*·In complex by gel filtration (koff,gel) or jump dilution (koff,jump). The E*·In complex was separated from excess inhibitor by gel filtration and then incubated with MnCl2 and PEP to prevent inhibitor rebinding, giving a koff,gel of 2.2 ± 0.2 h−1, corresponding to a residence time (tR = 1/ koff) of 27 min when [I]free ≈ 0 (Figure 7b). This was faster than kon,direct, which should not be possible, because that would mean that E*·In dissociates faster than it forms. In the jump dilution method, the E*·In complex was rapidly diluted 43-fold into buffer containing MnCl2 and PEP and gave a koff,jump of 0.09 ± 0.05 h−1, corresponding to a tR of 11 h, when [I]free = 47 μM (Figure 7c). Concentration Dependence of kobs. The concentration dependence of the rate of onset of inhibition, kobs, can be used to determine kon,conc and koff,conc.28 KDO8PSwt was incubated with varying KDO8P oxime concentrations for ≤25 h. Fitting initial velocities versus time for different inhibitor concentrations (eq 7, Figure S9) yielded kobs, which was then fitted versus KDO8P oxime concentration to yield a kon,conc of 0.23 ± 0.01 h−1 and a koff,conc of 0.014 ± 0.009 h−1, giving a tR of 69 h (eq 8, Figure 8). The value of kon,conc, 0.23 h−1, was lower than

Scheme 3. Kinetic Mechanism of Inhibition Showing (a) the Minimal Mechanism with Fast- and Slow-Binding Phasesa and (b) the Activation Model

a

The binding stoichiometries in part a are not known, so the generic labels E·In and E*·In are used.

kon,direct. The loss of activity of KDO8PSwt upon its preincubation with KDO8P oxime followed first-order kinetics, with a kon,direct of 0.36 ± 0.06 h−1 and a residual rate of 13% (Figure 7a). Direct koff Measurement. The rate of return of activity was measured directly after excess inhibitor was removed from

Figure 8. Concentration dependence of the rate of onset of slowbinding inhibition, kobs, of KDO8PSwt. The initial velocity vs time data were fitted to eq 7 to find kobs, which was then fitted vs KDO8P oxime concentration (eq 8). kon,conc = 0.23 ± 0.01 h−1, and koff,conc = 0.014 ± 0.009 h−1, with the Hill coefficient fixed at 3.5. The equivalent plot for KDO8PSH6 is shown in Figure S11.

kon,direct, 0.36 h−1, measured under slightly different conditions. The kon,direct value was expected to be higher because the assumption that k off = 0 becomes less valid as E*·I n accumulates, which has the effect of increasing kobs. The koff,conc value was not significantly different from zero, so it is not certain that any dissociation occurred under high-[I]free conditions. It was significantly lower than koff,gel and koff,jump, reinforcing the observation that koff depended on [I]free, with the following values: koff,gel = 2.2 h−1 ([I]free ≈ 0), koff,jump = 0.09 h−1 ([I]free = 47 μM), and koff,conc = 0.014 h−1 ([I]free = 100−10000 μM). The ultimate inhibition constant, Ki* (eq 10), was 0.57 ± 0.43 μM for KDO8PSwt. The main source of uncertainty was in the koff,conc of 0.014 ± 0.009 h−1, which was not statistically significantly different from zero, which could mean that the true Ki* value was lower than the reported value. The concentration dependence of kobs was also determined for KDO8PSH6 and yielded essentially the same koff,conc value, 0.016 ± 0.002 h−1 (Figures S10−S12). Concentration Dependence of Slow-Binding Inhibition. There was strong cooperativity in the concentration

Figure 7. Slow-binding inhibition of KDO8PSwt by KDO8P oxime. (a) kon,direct. v0/[E]0 vs preincubation time (eq 5). (b) koff,gel. The ratio of the initial velocities at times t and zero (vt/v0) vs time after gel filtration (eq 6). (c) koff,jump. vt/v0 vs time after jump dilution (eq 6). H

DOI: 10.1021/acs.biochem.8b00748 Biochemistry XXXX, XXX, XXX−XXX

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from the THI.16 KDO8P oxime’s behavior is consistent with this inhibitory mechanism. KDO8P oxime displayed residual activity. In the fast-binding phase, this could be due to negative cooperativity between inhibitor molecules bound to different subunits, leading to unoccupied subunits. It is also possible that there is positive cooperativity between the inhibitor and substrates bound to different subunits, as observed for DAHP oxime.15 The low residual rates implied 3.6−5.6-fold decreases in kcat for the unbound subunits, compared with 3-fold for DAHP oxime. Decreased activity in uninhibited subunits was clear evidence of intersubunit communication between bound and unbound subunits. Residual rates in the slow-binding phase, combined with the cooperativity of binding, implied that even though four bound inhibitor molecules were required to trigger isomerization to E*·In, only a subset became tightly bound. For example, the complex could be E*·I2,tight·I2,loose, though, because its composition was not known, it is simply labeled as E*·In. The increase in koff as [I]free decreased implied that these weakly bound inhibitor molecules helped to stabilize the tightly bound sites in E*·In. The concentration dependence of KDO8P oxime’s slowbinding inhibition had a Hill coefficient (n) of 3.5 for KDO8PSwt, compared with a value of 3.9 for DAHP oxime with DAHPS,15 suggesting a common mechanism. This was in apparent contradiction with the lack of cooperativity in fastbinding inhibition, but it was consistent with an “activation” model54 in which isomerization of E·In to E*·In is activated only once all active sites are occupied (Scheme 3b). Multiple binding sites, in themselves, do not cause strong cooperativity; four independent binding sites with equal Kd values would have an n of 1.3.54 Strong cooperativity requires either an activation model, which was consistent with the experimental data, or decreasing Kd’s as the number of bound ligands increases, which was not found. The ultimate inhibition constant (Ki*) with KDO8PSwt was 0.57 μM, making KDO8P oxime the second-tightest-binding KDO8PS inhibitor characterized to date,11 though with the caveat that tight binding is maintained only in the presence of excess KDO8P oxime. Mode of Inhibition with Respect to Metal Ions. KDO8P oxime and DAHP oxime had different modes of inhibition with respect to Mn2+. DAHP oxime bound competitively with respect to Mn2+ even though it did not occupy the same physical space in the active site and did not disrupt any of the metal ion-binding side chains.15 In contrast, KDO8P oxime binding was enhanced by Mn2+, making its mode of inhibition uncompetitive-like with respect to Mn2+. Its Kd decreased at least 10-fold in the presence of Mn2+, corresponding to a 1.4 kcal/mol increase in inhibitor binding. The reasons for competitive versus uncompetitive binding are not known for either enzyme; however, there are significant differences in how each enzyme interacts with its metal ion, which could lead to different modes of inhibition. Specifically, all known DAHPSs are metal-dependent, with the metal ion interacting with the E4P carbonyl oxygen to polarize the aldehyde functional group for nucleophilic attack by PEP’s C3. In contrast, some KDO8PSs are metal-independent,4,36,46 and interconversion between metal dependence and independence is facile.45−48 A5P’s carbonyl oxygen is proposed to interact with PEP’s phosphate group in KDO8PS, and there is no clear evidence that the metal ion has a catalytic, as distinct from structural, role.55

dependence of slow-binding inhibition (Figure 9). The IC50 value was larger than Ki for fast-binding inhibition, as expected

Figure 9. Concentration dependence of slow-binding KDO8PSwt inhibition after preincubation for 25 h. Initial velocities were fitted to eq 9. IC50 = 3100 ± 200 μM, and n = 3.5 ± 0.5. Inhibitor concentrations are those in the preincubation mixture, before dilution into the assay mixture. The equivalent plot for KDO8PSH6 is shown in Figure S12.

for preincubation in the absence of Mn2+. With KDO8PSwt, IC50 = 3100 ± 200 μM, with a Hill coefficient, n, of 3.5 ± 0.5. The Hill coefficient indicated that multiple inhibitor molecules are needed to induce slow-binding inhibition. However, there was an ≈15% residual rate, implying that the tight-binding complex was induced in only some of the subunits, with the remaining subunits remaining in the non-isomerized, weakly bound, form that could be displaced by substrates in the rate assays.

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DISCUSSION KDO8PS Characterization. KDO8PS’s kinetic mechanism was metal-dependent, rapid equilibrium sequential ordered ter ter, with Mn2+ binding first, then PEP, and then A5P. 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. The rapid equilibrium assumption, though, will not always be valid. KDO8PS and E. coli DAHPS(Phe)15both required treatment with EDTA for 24−48 h to remove endogenous metal ions after purification, and the freshly purified enzyme was pinkish, as was A. aeolicus KDO8PS,34,52 presumably because of bound Fe2+. The steady-state approximation will be necessary in cases in which metal dissociation is slow. KDO8P Oxime Inhibition. KDO8P oxime was based on DAHP oxime, a DAHPS inhibitor with a fast-binding Ki of 1.5 μM and a residence time of 83 min.15 The oxime group, combined with two crystallographic waters, structurally mimics the PEP/THI phosphate group. The nitrogen atom occupies the same location as the phosphate group’s bridging oxygen and hydrogen bonds to the presumed general acid catalyst, K186.53 The same interaction is possible between KDO8P oxime and K120, the positional homologue of K186. Linear free energy relationship (LFER) analysis showed that DAHP oxime is a transition-state mimic inhibitor, presumably mimicking the transition state for the departure of phosphate I

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Residence Time. KDO8P oxime had a residence time of up to almost 3 days. There is a growing body of evidence that drugs’ in vivo efficacy is closely related to their residence times in their target enzyme’s active sites.17,56 For example, Efavirenz (Ki = 5 μM, and tR = 4 h)57 effectively treats HIV in spite of its high Ki value, presumably because of its long residence time. The fact that DAHP oxime and KDO8P oxime both had significant residence times implies that this may be a consistent feature of this class of inhibitors. α-Carboxyketose Synthase Inhibition. KDO8P oxime helped confirm the generality of the oxime functional group as an inhibitory motif for α-carboxyketose synthases. Given its structural and functional mimicry of a phosphate group, it functions as a small, neutral phosphate bioisostere,58 even though it is not an isostere in the chemical sense. In the same way, carboxylates are considered phosphate bioisosteres even though they are not chemical isosteres.59 It illustrated both the promise and the challenges of inhibiting this enzyme superfamily. DAHP oxime appears to mimic the transition state of phosphate departure during THI breakdown.16 All αcarboxyketose synthases should be susceptible to this inhibitory mechanism as long as THI breakdown is kinetically significant. “Kinetically significant” means being the first (fully) irreversible step and is reflected in the specificity constant, kcat/ (KM,MnKM,PEPKM,aldose).16,60 KDO8P oxime’s effectiveness demonstrated that THI breakdown is kinetically significant for KDO8PS and therefore amenable to further refinement of the inhibitory motif. It is also possible that THI formation is also partially irreversible, which would make both steps legitimate targets for inhibition. The residual rates at high KDO8P oxime concentrations and the dependence of the residence time on free inhibitor concentration illustrate the challenges of designing inhibitors of oligomeric enzymes that display intersubunit interactions. Residual rates were also observed with DAHP oxime, as well as positive cooperativity between inhibitor binding and substrate binding to the unoccupied subunits, which implies that there could be some combination of structural features that would lead to positive cooperativity of inhibitor binding with suitably designed inhibitors.

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Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.8b00748. Primer and enzyme sequences, sequence alignments, SDS−PAGE gels, purification scheme, random order and steady-state ordered kinetic mechanisms, KDO8PS oligomer structure, KDO8PSH6 kinetic parameters and inhibition constant, mode of inhibition with respect to PEP, and rate of onset of slow-binding inhibition (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Paul J. Berti: 0000-0003-3409-7907 Funding

This work was supported by Canadian Institutes of Health Research Operating Grant MOP-64422 (P.J.B.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Martin Young (National Research Council of Canada) for the generous gift of the C-terminally tagged KDO8PS plasmid and Dr. José Carlos Bozelli Jr. and Dr. Richard Epand for help with ITC experiments.

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ABBREVIATIONS A5P, arabinose 5-phosphate; BSA, bovine serum albumin; BTP-HCl, bis-tris propane hydrochloride; K-CAPS, potassium N-cyclohexyl-3-aminopropanesulfonate; AHP, 3-deoxy-D-arabino-heptulosonate 7-phosphate; DAHPS, DAHP synthase; E4P, erythrose 4-phosphate; ITC, isothermal titration calorimetry; KDO8P, 3-deoxy-D-manno-2-octulosonate 8phosphate; KDO8PS, KDO8P synthase; KDO8PSH6, C. jejuni KDO8PS with an N-terminal His6-TEV tag; KDO8PSwt, wild type C. jejuni KDO8PS; LB, lysogeny broth; MG/AM, Malachite green/ammonium molybdate; PEP, phosphoenolpyruvate; Pi, inorganic phosphate; TCEP, tris(2-carboxyethyl)phosphine.

CONCLUSIONS

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C. jejuni KDO8PS is an antimicrobial target. It is a metaldependent homotetrameric enzyme that follows a rapid equilibrium sequential ordered ter ter kinetic mechanism. KDO8P oxime is an attractive inhibitor, showing fast-binding inhibition, with a Ki of 10 μM, and slow-binding inhibition with an ultimate inhibition constant (Ki*) down to 0.57 μM, and residence times of up to 69 h in the presence of excess inhibitor. The cooperativity of slow-binding inhibition implied that it occurs only when all four active sites are occupied. Its dissociation rate constant, koff, depended on the free inhibitor concentration, with residence times of 27 min to 69 h. This implied intersubunit communication between the tightly bound and loosely bound subunits and, as with DAHP oxime inhibition of DAHPS, showed that effective inhibition of these enzymes will require understanding how both substrate binding and inhibitor binding are modulated by intersubunit communication.

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DOI: 10.1021/acs.biochem.8b00748 Biochemistry XXXX, XXX, XXX−XXX