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

Helene J Bustad; Juha P Kallio; Mikko Laitaoja; Karen Toska; Inari Kursula; Aurora Martinez; Janne Jänis

doi:10.1016/j.isci.2021.102152

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

iScience. 2021 Feb 6;24(3):102152. doi: 10.1016/j.isci.2021.102152. eCollection 2021 Mar 19.

Authors

Helene J Bustad¹, Juha P Kallio¹, Mikko Laitaoja², Karen Toska³, Inari Kursula^{1

4}, Aurora Martinez¹, Janne Jänis²

Affiliations

¹ Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway.
² Department of Chemistry, University of Eastern Finland, 80130 Joensuu, Finland.
³ Norwegian Porphyria Centre (NAPOS), Department for Medical Biochemistry and Pharmacology, Haukeland University Hospital, 5021 Bergen, Norway.
⁴ Faculty of Biochemistry and Molecular Medicine, University of Oulu, 90570 Oulu, Finland.

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.