Structural basis of pyrrole polymerization in human porphobilinogen deaminase

Abstract

Human porphobilinogen deaminase (PBGD), the third enzyme in the heme pathway, catalyzes four times a single reaction to convert porphobilinogen into hydroxymethylbilane. Remarkably, PBGD employs a single active site during the process, with a distinct yet chemically equivalent bond formed each time. The four intermediate complexes of the enzyme have been biochemically validated and they can be isolated but they have never been structurally characterized other than the apo- and holo-enzyme bound to the cofactor. We present crystal structures for two human PBGD intermediates: PBGD loaded with the cofactor and with the reaction intermediate containing two additional substrate pyrrole rings. These results, combined with SAXS and NMR experiments, allow us to propose a mechanism for the reaction progression that requires less structural rearrangements than previously suggested: the enzyme slides a flexible loop over the growing-product active site cavity. The structures and the mechanism proposed for this essential reaction explain how a set of missense mutations result in acute intermittent porphyria.

Keywords: Acute intermittent porphyria; Enzyme mechanism; Heme biosynthesis; Human porphobilinogen deaminase (PBGD); Reaction intermediates; X-ray structure.

Figure 1

Native polyacrylamide electrophoresis for the different reaction intermediates isolated for human PBGD. The band downshift suggests a gradual increase in net negative charge as compared to E_apo. ES₂ and ES₂' both correspond to the reaction intermediate with two PBG condensed units. The presence of multiple bands probably indicates incomplete separation between the intermediates. The asterisk indicates the intermediate that was successfully crystallized.

Figure 2

Biophysical properties of the PBGD reaction intermediates. (A) Superimposition of the relaxed model structure (3ECR, after 17 ns MD simulation) with the SAXS-derived low-resolution protein volumes. (B) Fitting of the experimental scattering spectra with a simulated fit obtained from the relaxed structure. (C) ¹H,¹⁵N-HSQC spectra for the isolated PBGD reaction intermediates show chemical shift perturbations consistent with localised structural changes.

Figure 3

(A) Overall structure of human PBGD. Cartoon representation of human PBGD bound with dipyromethane cofactor (E_holo, erythrocytic PBGD isoform, green) overlaid with the reaction intermediate (ES₂, housekeeping isoform, blue). The arrow indicates the likely pathway for a new PBG unit as well as the direction of the loop displacement. (B) Root mean square deviation values between the residues of E_holo (reference) and ES₂-complex structures. Values below the average (r.m.s.d_av = 0.434) covered with grey color. (C) Cartoon representation of ES₂ PBGD showing the loop region covering the active site, red highlighted. The two orientations correspond to a 90º rotation along the ordinate reference axis.

Figure 4

Active site of human PBGD. (A-B) Electron density for the cofactor/ligand in E_holo and ES₂. The interactions between the cofactor or the reaction intermediate and the residues from the active site are shown in (C) and (D) for E_holo and ES₂, respectively. The cavity volume enclosing the active site is shown in (E) and (F) for E_holo and ES₂. For clarity, the cavity has been divided into the frontal (purple) and rear (orange) sub-cavities, as indicated. Distances between the catalytic residue D99 and some atoms relevant for the reaction are shown in (G) and (H) for E_holo and ES₂-complex.

Figure 5

Scheme showing the active site cavity as a function of the reaction progression. The R255-V263 loop hosts C261 which covalently binds the cofactor. The loop displaces during polypyrrole elongation. At all times, D99 is at reactive distance of the S2 and C2 positions in the ES₂ complex. According to our model, once the ES₄ complex is reached, the hydrolysis occurs spontaneously and mediated by D99 (reverse reaction), as indicated. The cavities for E_holo and ES₂ (dash gray line highlight) are depicted from the high-resolution structures while the other cavities are theoretical models.