Crystal structures of hydroxymethylbilane synthase complexed with a substrate analog: a single substrate-binding site for four consecutive condensation steps

Abstract

Hydroxymethylbilane synthase (HMBS), which is involved in the heme biosynthesis pathway, has a dipyrromethane cofactor and combines four porphobilinogen (PBG) molecules to form a linear tetrapyrrole, hydroxymethylbilane. Enzyme kinetic study of human HMBS using a PBG-derivative, 2-iodoporphobilinogen (2-I-PBG), exhibited noncompetitive inhibition with the inhibition constant being 5.4 ± 0.3 µM. To elucidate the reaction mechanism of HMBS in detail, crystal structure analysis of 2-I-PBG-bound holo-HMBS and its reaction intermediate possessing two PBG molecules (ES2), and inhibitor-free ES2 was performed at 2.40, 2.31, and 1.79 Å resolution, respectively. Their overall structures are similar to that of inhibitor-free holo-HMBS, and the differences are limited near the active site. In both 2-I-PBG-bound structures, 2-I-PBG is located near the terminus of the cofactor or the tetrapyrrole chain. The propionate group of 2-I-PBG interacts with the side chain of Arg173, and its acetate group is associated with the side chains of Arg26 and Ser28. Furthermore, the aminomethyl group and pyrrole nitrogen of 2-I-PBG form hydrogen bonds with the side chains of Gln34 and Asp99, respectively. These amino acid residues form a single substrate-binding site, where each of the four PBG molecules covalently binds to the cofactor (or oligopyrrole chain) consecutively, ultimately forming a hexapyrrole chain. Molecular dynamics simulation of the ES2 intermediate suggested that the thermal fluctuation of the lid and cofactor-binding loops causes substrate recruitment and oligopyrrole chain shift needed for consecutive condensation. Finally, the hexapyrrole chain is hydrolyzed self-catalytically to produce hydroxymethylbilane.

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

“…This suggests that the role of this loop is incidental to the catalytic sequence of events, at least up to ES 2 . However, Sato et al (2021) report that 'flexibility of this loop in the proximity of the active site appears to be involved in the binding of 2-I-PBG and the substrate, although no direct interactions between the loop (residues 58-69) and 2-I-PBG were observed'. This last sentence seems to be a little self-contradictory.…”

“…They also reported that 'The overall structure of the 2-I-PBG-bound holo-HMBS was found to be similar to that of the inhibitor-free holo-HMBS'. Table 2 of Sato et al (2021) provides comprehensive details of the interactions between pyrroles and the protein moiety in HMBS (in their four crystal structures). Fig.…”

“…Fig. 8 of Sato et al (2021) shows a predicted ES 1 based on their crystal structure of human HMBS complexed with 2-I-PBG. As a historical note, bromoporphobilinogen was important in the study of the enzyme, as experiments by Warren & Jordan (1988) using bromoporphobilinogen provided conclusive evidence for the direct covalent interaction of the substrate with the dipyrromethane (DPM) cofactor.…”

“…However, if the mass is directly measured then this datum assumes a special importance. This approach of measuring the mass was also performed by Sato et al (2021).…”

Combining X-rays, neutrons and electrons, and NMR, for precision and accuracy in structure–function studies

“…This was a significant, complementary observation to the electron density map changes in the enzyme active site. Subsequent to these experiments the human HMBS enzyme has been studied by static X-ray crystallography in the apo and ES2 forms by trapping of this state (Pluta et al, 2018;Kallio et al, 2021;Sato et al, 2021). The two approaches, time-resolved and static crystallography, yield complementary views of the intermediate structures, ES2.…”