Allosteric feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia coli

Abstract

In Escherichia coli, the synthesis of pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B₆, takes place through the so-called deoxyxylulose 5-phosphate-dependent pathway, whose last step is pyridoxine 5'-phosphate (PNP) oxidation to PLP, catalyzed by the FMN-dependent enzyme PNP oxidase (PNPOx). This enzyme plays a pivotal role in controlling intracellular homeostasis and bioavailability of PLP. PNPOx has been proposed to undergo product inhibition resulting from PLP binding at the active site. PLP has also been reported to bind tightly at a secondary site, apparently without causing PNPOx inhibition. The possible location of this secondary site has been indicated by crystallographic studies as two symmetric surface pockets present on the PNPOx homodimer, but this site has never been verified by other experimental means. Here, we demonstrate, through kinetic measurements, that PLP inhibition is actually of a mixed-type nature and results from binding of this vitamer at an allosteric site. This interpretation was confirmed by the characterization of a mutated PNPOx form, in which substrate binding at the active site is heavily hampered but PLP binding is preserved. Structural and functional connections between the active site and the allosteric site were indicated by equilibrium binding experiments, which revealed different PLP-binding stoichiometries with WT and mutant PNPOx forms. These observations open up new horizons on the mechanisms that regulate E. coli PNPOx, which may have commonalities with the mechanisms regulating human PNPOx, whose crucial role in vitamin B₆ metabolism and epilepsy is well-known.

Keywords: Escherichia coli; allosteric inhibition; allosteric regulation; enzyme mechanism; linear mixed-type inhibition; pyridoxal 5'-phosphate; pyridoxal phosphate; pyridoxine 5'-phosphate oxidase; vitamin; vitamin B6 biosynthesis.

Figure 1.

Kinetics of PNP oxidation to PLP in Tris and HEPES buffers. Shown is a comparison of kinetics obtained with 0.5 μm enzyme (protein subunit concentration) and 15 μm PNP, carried out in 50 mm Tris-HCl and 50 mm NaHEPES buffers at pH 7.6. Reactions, carried out at 37 °C, were started by the addition of the enzyme to buffer containing PNP and kept under constant stirring by a magnetic bar, to ensure a rapid mixing. The kinetics trace obtained in Tris buffer was fitted to a closed form of the time-integrated Michaelis–Menten equation (21), whereas the time course of the reaction carried out in HEPES buffer was fitted to Equation 1 (continuous black lines through the experimental traces). The inset shows the first derivative of experimental and theoretical traces. Kinetic traces were exactly the same when the order of addition of reaction components was inverted by adding PNP last.

Figure 2.

Analysis of reactions catalyzed by PNPOx in HEPES buffer. All reactions were carried out in 50 mm NaHEPES buffer, pH 7.6, as explained in the legend to Fig. 1. A, a fixed concentration of enzyme (0.5 μm) was mixed with buffer containing different PNP concentrations (1.88, 3.75, 7.5, 15, 30, 60, and 100 μm). Starting reactions by the addition of substrate as the last component yielded identical kinetics (data not shown). All kinetics were fitted to Equation 1. The inset shows the concentration of PLP formed in the deceleration phase, as determined from fitting, as a function of substrate concentration. The continuous line describes the trend of the experimental points and has no analytical purpose. Values of k_E and k_L parameters found in the fitting are shown in Fig. S1. B, the saturation curve obtained by plotting the initial velocity of the reaction as a function of substrate concentration was analyzed using quadratic Equation 2, obtaining the kinetic parameters reported in Table 1. Error bars, S.E. of the initial velocity values estimated in the fitting procedure. C, increasing enzyme concentration (0.25, 0.5, 0.75, 1, and 2 μm), while keeping PNP concentration fixed (15 μm), proportionally increased the amount of PLP produced in the deceleration phase, as shown in the inset and determined from fitting of kinetics to Equation 1. D, kinetics obtained by the addition of increasing concentrations of exogenous PLP (0, 0.25, 0.5, 1, 2, 4, 8, and 16 μm) to reactions containing 0.5 μm enzyme and 15 μm PNP. Reactions were started by adding PNP as the last component to solutions of enzyme and PLP. However, the order of addition of reactants did not affect kinetics, demonstrating that both PNP and PLP bind rapidly to the enzyme. Fitting to Equation 1 gives the amplitude of the deceleration phase as a function of exogenous PLP concentration (inset). The continuous line has the purpose of describing the trend of experimental points.

Figure 3.

Characterization of PLP inhibition. A, the initial velocity of the reaction was measured with 0.5 μm enzyme (protein subunit concentration), varying PNP concentration while keeping exogenous PLP fixed and at different concentrations (0, 0.25, 0.5, 1, 2, 4, 8, and 16 μm). The obtained saturation curves were fitted to Equation 2, obtaining estimates of apparent k_cat and K_D. B, fitting of apparent k_cat (blue symbols) and K_D (red symbols), using Equations 3–5, as explained under “Experimental procedures,” gave estimates of dissociation and inhibition constants, which are reported in Table 1. Error bars, S.E. of parameter values estimated in the fitting procedure. Concentration of PLP on x axes is the total concentration.

Scheme 1.

A, steady-state kinetics scheme describing a linear mixed-type inhibition system, in which the enzyme (E) is able to bind both the PNP substrate (S) and the PLP product (P) at the same time. B, ping-pong kinetic mechanism of the reaction catalyzed by PNPOx, in which E_o and E_r are the oxidized form and reduced form of the enzyme, respectively. The PLP product inhibits the enzyme when it binds to the E_o form.

Figure 4.

Analysis of PLP-binding equilibrium. Emission spectra (from 470 and 570 nm) of PNPOx in the presence of different PLP concentrations were measured in 50 mm NaHEPES, pH 7.6, upon excitation at 450 nm. The figure shows the PLP-binding curve obtained with 100 nm PNPOx (protein subunit concentration). The average relative fluorescence emission between 520 and 530 nm (F_r(520–530)) as a function of total PLP concentration was analyzed with a quadratic equation describing the binding of a ligand at a single site (Equation 6), using the value of 100 nm protein subunit concentration as a fixed parameter in the fitting procedure. A dissociation constant of 147 ± 43 nm was calculated from the analysis of five independent experiments, such as that shown in the figure. Inset, binding stoichiometry analysis obtained with three different and much higher protein subunit concentrations (1 μm (black symbols), 2 μm (red symbols), and 4 μm (blue symbols)). Fluorescence change, expressed as fractional variation as a function of the [PLP_tot]/[protein] ratio, is linear as shown by the thick continuous line, up to the stoichiometry point corresponding to the crossing with the horizontal dotted line. The vertical thin line through the stoichiometry point indicates that about one PLP molecule binds per enzyme dimer.

Figure 5.

PLP binding as measured by stopped-flow spectroscopy. 20 μm PNPOx (protein subunit concentration) was mixed 1:1 in the stopped-flow apparatus with PLP (2, 4, 6, 8, 13, 15, 24, 25, 29, 30, 33, and 37 μm) in NaHEPES buffer at 25 °C. A, for a better view, the time courses of the reactions were offset by 0.1 fluorescence units on the y axes by adding an increasing value and are shown on a log(time) scale. Traces were fitted to Equation 7 (solid lines). B, dependence of k_fast (k_f; blue symbols) and k_slow (k_s; red symbols) on PLP concentration after mixing; k_fast was fitted to Equation 8, and k_slow was fitted to a line with slope = 0 (solid lines).

Scheme 2.

Kinetic mechanism of PLP binding to PNPOx. See “Results” for a description of this mechanism.

Figure 6.

PLP binding to enzyme forms with altered active site structure. Fluorometric analysis of PLP binding to apo-PNPOx (100 nm, protein subunit concentration) was carried out exciting at 280 nm. The average fluorescence emission between 335 and 345 nm was plotted against total PLP concentration (black symbols). PLP binding to the holo-form of the quadruple mutant (red symbols) was analyzed as described previously for WT holo-form PNPOx. Both sets of data were fitted using the quadratic Equation 6, obtaining K_D values reported in Table 1. Inset, binding stoichiometry analysis obtained with three different and much higher concentrations of the quadruple PNPOx mutant, as explained in Fig. 4 for the WT enzyme. Protein subunit concentrations were 4 μm (black symbols), 8 μm (red symbols), and 12 μm (blue symbols).

Figure 7.

Enlarged view of PNPOx active site structure. Cartoon backbone representation of the E. coli PNPOx dimer (Protein Data Bank code 1G79) with PLP bound at the active site. The two subunits are shown in cyan and salmon, respectively. Active site residues involved in binding of PLP are shown as sticks and labeled. Interatomic distances indicated by red dashes are expressed in Å. It can be assumed that the PNP substrate binds by establishing the same interactions with active-site amino acid residues (23).

Figure 8.

Kinetics of PNP oxidation to PLP in HEPES buffer catalyzed by WT PNPOx with tightly bound PLP. Reactions were carried out as explained in the legend to Fig. 1 and started by diluting enzyme samples, either the PNPOx-PLP complex obtained after incubation with PLP and size-exclusion chromatography (red trace) or an untreated control PNPOx sample (black trace) in 50 mm NaHEPES buffer, pH 7.6, containing PNP, obtaining 8 μm enzyme and 240 μm substrate final concentrations.