Figure 1.
Organization of the OAD complex.
a. Structural model and catalytic events of the oxaloacetate decarboxylase. Oxaloacetate decarboxylase is a membrane-bound enzyme complex composed of α, β, and γ subunits in a 1∶1∶1 molar ratio. The α subunit is soluble and harbors the carboxyltransferase catalytic site. The carboxyl group from position 4 of oxaloacetate is transferred to the biotin prosthetic group bound to the C-terminal biotin-binding domain. The carboxybiotin formed switches to the decarboxylase site on subunit β, where decarboxylation takes place and free biotin is regenerated, using one periplasmic proton. During the reaction, two sodium ions are translocated from the cytoplasm into the periplasm. Adapted from [9]. b. Structure of the carboxyltransferase domain of OAD α subunit highlighting the position of tryptophan residues. Four of them (positions 45, 50, 67, 160) are located within the catalytic α8β8 subdomain whereas a fifth tryptophan (W448) is located in the noncatalytic subdomain. This figure was drawn using YASARA (www.yasara.org) from PDB file 2NX9.
Figure 2.
Fluorescence emission spectrum at 295 nm excitation wavelength of OAD in the absence (black circles) or presence (gray triangles) of 10 mM Oxomalonate.
Each spectrum is the average of at least three determinations. Samples were in 250 mM NaCl, 0.5% Tween 20, 0.05% Brij 58 50 mM, Tris-HCl, pH 7.4 buffer.
Figure 3.
Red edge excitation shift observed as a result of oxomalonate binding to Oxaloacetate Decarboxylase.
Maximum wavelength of the emission spectra at different excitation wavelengths of OAD in the absence (black) or presence (gray) of 10 mM Oxomalonate. Each point is the average of at least three determinations. White and gray bars (right) correspond to OAD REES observed in the absence and in the presence of oxomalonate respectively. Samples were in 250 mM NaCl, 0.5% Tween 20, 0.05% Brij 58, 50 mM Tris-HCl, pH 7.4 buffer.
Figure 4.
Effects of oxomalonate binding on the tertiary structure of OAD α subunit.
REES variations observed on the nonbiotinylated (a, c) or biotinylated (b, d) α subunit in absence (a, b) or presence (c, d) of 10 mM oxomalonate. Each point is the average of at least three determinations. Samples were in 250 mM NaCl, 0.5% Tween 20, 0.05% Brij 58, 50 mM Tris-HCl, pH 7.4 buffer.
Figure 5.
Red edge excitation shift observation as a result of the oxomalonate effect on the OAD αγ subunits structure.
A. Maximum wavelength of the emission spectra of OAD α subunit at increasing excitation wavelengths in the absence (black rectangles) or presence (white rectangles) of 10 mM Oxomalonate. B. REES observed on the αγ subunit spectrum in the absence (black) or presence (white rectangles) of 10 mM Oxomalonate when the excitation was shifted from 275 nm to 307 nm. Each point is the average of at least three determinations. Samples were in 250 mM NaCl, 0.5% Tween 20, 0.05% Brij 58 50 mM, Tris-HCl, pH 7.4 buffer.
Figure 6.
Influence of substrate binding on OAD and OAD subunits.
A. OAD REES variations in the absence of substrates (a) or in the presence of either 250 mM NaCl (b) or 10 mM oxomalonate (c). OAD was purified in 250 mM KCl, pH 7.4, 0.5% Tween 20, 0.05% Brij 58, 100 mM KH2PO4 buffer. REES of OAD in KCl-containing buffer (i.e. 3.8 nm, not shown) was taken as reference to calculate the REES variation. B. Na+ (250 mM) influence on REES variation of carboxyltransferase domain of the α subunit (a), reconstituted carboxyltransferase and biotin binding domain of the α subunit (b), the whole α subunit (c), αγ complex (d), and the OAD complex (e).
Figure 7.
Amide I comparative secondary structure spectra of OAD and OAD subunits.
OAD (a), αγ (b), biotinylated α (c), and nonbiotinylated α (d) subunits.
Figure 8.
Effect of oxomalonate binding on OAD and OAD subunits secondary structure.
(a) OAD in presence of NaCl, (b) αγ subunit, (c) α subunit, (d) biotinylated α subunit. Full and dotted lines represent the complexes without and with oxomalonate respectively.
Figure 9.
Na+ influence on OAD secondary structure and on the ability to bind oxomalonate.
a. OAD secondary structure in the absence of NaCl (red line) and after the addition of 250 mM NaCl (blue line). b. OAD secondary structure in the absence NaCl before (red full line) and after addition of 10 µM oxomalonate (black dotted line). OAD was purified in 250 mM KCl, pH 7.4, 0.5% Tween 20, 0.05% Brij 58, 100 mM KH2PO4 buffer.