Characterization of porphobilinogen deaminase mutants reveals that arginine-173 is crucial for polypyrrole elongation mechanism

Abstract

Porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthesis, catalyzes the sequential coupling of four porphobilinogen (PBG) molecules into a heme precursor. Mutations in PBGD are associated with acute intermittent porphyria (AIP), a rare metabolic disorder. We used Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to demonstrate that wild-type PBGD and AIP-associated mutant R167W both existed as holoenzymes (E_holo) covalently attached to the dipyrromethane cofactor, and three intermediate complexes, ES, ES₂, and ES₃, where S represents PBG. In contrast, only ES₂ was detected in AIP-associated mutant R173W, indicating that the formation of ES₃ is inhibited. The R173W crystal structure in the ES₂-state revealed major rearrangements of the loops around the active site, compared to wild-type PBGD in the E_holo-state. These results contribute to elucidating the structural pathogenesis of two common AIP-associated mutations and reveal the important structural role of Arg173 in the polypyrrole elongation mechanism.

Keywords: Biochemistry; Biological Sciences; Proteomics; Structural Biology.

Graphical abstract

Figure 1

Crystal structure of hPBGD wt-E_holo and schematic representation of the polypyrrole elongation mechanism (A) Overall representation of the crystal structure of wt-hPBGD (PDB: 7AAJ), providing the expected holoenzyme (E_holo) with three separate domains (domain 1 is presented in red, domain 2 in light brown, and domain 3 in green). The bound dipyrromethane (DPM) cofactor is shown in a brown stick representation. (B) Schematic representation of the general understanding of the mechanism of a single step in the polypyrrole elongation. (C) The polypyrrole elongation catalyzed by PBGD. PBGD with attached DPM (E_holo) subsequently binds four porphobilinogen (PBG) substrates (S), and generates the enzyme intermediates ES, ES₂, ES₃, and ES₄. The linear product, hydroxymethylbilane (HMB), is released by hydrolysis and cyclized by the next enzyme in the heme biosynthesis. The sidechains of the substrates, acetate (CH₂CO₂H) and propionate (CH₂CH₂CO₂H) are denoted Ac and Pr, respectively. Figure 1C is modified from (Jordan and Woodcock, 1991).

Figure 2

High-resolution mass spectrometry of hPBGD enzymes The ESI FT-ICR mass spectra were measured at denaturing conditions with 5 μM protein. (A) Broadband mass spectrum of wt-hPBGD with numbers denoting different protein ion-charge states. A wide charge state distribution from 18 + to 45+ is consistent with the protein being fully unfolded. (B) Charge-deconvoluted mass spectrum showing peaks representing different enzyme-intermediates in wt-hPBGD. (C) and (D) Charge-deconvoluted mass spectra of the hPBGD mutants R167W and R173W, respectively. The peaks representing different enzyme-intermediates are assigned. See also Figures S1–S3, and Tables S1 and S2.

Figure 3

The crystal structure of hPBGD wt-E_holo and R173W-ES₂ (A) Overall cartoon representation of wt-E_holo (gray; PDB: 7AAJ) and mutant R173W-ES₂ (blue; PDB: 7AAK) superimposed. DPM cofactor with C1 and C2 units of wt-E_holo is shown in brown and elongation product of R173W-ES₂ including S1 and S2 units is shown in green. The mutated residue studied here, R173W, is shown as sticks. The cofactor-binding loop and cofactor are rearranged (red arrow) in the structure, allowing incoming substrate pyrroles (S1 and S2) substitute C1 and C2 at equal positions as in the E_holo. (B) The active-site loop (residues 57–74) orientation in wt-ES₂ (PDB: 5M6R; dark gray (Pluta et al., 2018)) is compared to the loop orientation in R173W-ES₂ (red). Formed ⍺2₁ helixes are labeled. Close-up also shows the movement of the cofactor-binding loop (orange; residues 257–263), and the elongation product in the R173W mutant. The position of cofactor-binding Cys261 has been indicated with labels C261-E_holo and C261-ES₂ for wt-E_holo and R173W-ES₂ structures, respectively. Glycerol (GOL) partially filling the solvent cavity under the cofactor-binding loop in the R173W-ES₂ structure is shown as sticks (yellow). See also Figures S4 and S5.

Figure 4

Electron density for the structural features Calculated 2mFo-DFc-electron density for (A) the active-site loop in R173W-ES₂, (B) bound cofactor in wt-E_holo and (C) the polypyrrole chain in R173W-ES₂. Electron density is contoured with sigma level of 1.0.

Figure 5

Stick representation of the active site The image shows active site interactions of (A) our wt-E_holo (side chains gray and substrate brown), and (B) R173W-ES₂ (side chains blue and substrate green) superimposed with wt-ES₂ (PDB: 5M6R; side chains dark gray and substrate pink (Pluta et al., 2018)). Incoming PBG units in the ES₂ intermediates substitute C1 and C2 at equal positions as in the E_holo. Interactions are described in detail in Table 2.

Figure 6

Schematic view of the interactions between the pyrrole rings and the hPBGD protein in the crystal structures of wt-E_holo and R173W-ES₂ mutant (A) The hydrogen bond interactions of the DPM cofactor with the wt-hPBGD residues in the active site. (B) The interactions between the pyrrole chain intermediate as seen in the crystal structure of the R173W-hPBGD mutant. Side chain H-bond interactions (blue), H-bond interactions to main chain carbonyl oxygen (green) and H-bond interactions to nitrogen (red).