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

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, ES(2), and ES(3), where S represents PBG. In contrast, only ES(2) was detected in AIP-associated mutant R173W, indicating that the formation of ES(3) is inhibited. The R173W crystal structure in the ES(2)-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.