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

Abstract

Summary 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.

The crystal structures of the enzyme hydroxymethylbilane synthase, also known as porphobilinogen deaminase

“…This last sentence seems to be a little self-contradictory. not produced, then it is compelling to agree with Bustad et al (2021) that 'the substrate elongation from ES 2 to ES 3 is crucially dependent on Arg173'. Secondly, since Pluta et al (2018) actually crystallized ES 2 for the wild-type enzyme and Fig.…”

“…How do we reconcile these observations with the agreed model (Figs. 3, The motion of (human) Cys261 itself is largely responsible for pulling the cofactor to make room for the addition of two PBG molecules to form ES 2 (Bustad et al, 2021). The movement of Cys261 (PDB entries 7aaj and 7aak, molecules A) is 4.4 A ˚and that of Val263 is 0.8 A ˚.…”

“…4.6. Consistency given by combining different methods Bustad et al (2021) elegantly show how high-resolution mass spectrometry allows direct analysis of the intermediates in order to directly plan the X-ray crystal structures. A weakness of an X-ray crystal structure is that where there is disorder, such as a mobile loop or a floppy tripyrrole or tetrapyrrole, there will be broken up or missing electron density.…”

“…These suggested that thermal fluctuation of the lid (residues 58-75, human numbering) and cofactor-binding loops causes the substrate recruitment and oligopyrrole chain shift needed for consecutive condensation. This suggestion relates to the observation by Bustad et al (2021) that Cys261 is the locus of the movement of the cofactor during catalysis, but what is the 'engine' or energy to drive this movement? Perhaps Sato et al (2021) have the answer, namely thermal fluctuation (of the lid and cofactor-binding loops).…”

Characterisation of a common hotspot variant in acute intermittent porphyria sheds light on the mechanism of hydroxymethylbilane synthase function

“…Recent advances in the field include the crystal structure of wt-HMBS in its ES 2 -state (PDB ID: 5M6R), demonstrating how the cofactor-binding loop with the Cys261 residue that anchors the cofactor, is pulled backwards with the two incoming PBG substrates [11]. The two new substrate molecules replace the cofactor in their positions and interactions [11,52]. Furthermore, our report on the p.R173W-variant trapped in the ES 2 -state (PDB ID: 7AAK) revealed that Arg173 is an essential residue for the enzyme to move from ES 2 to ES 3 in the elongation process [52].…”