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

Hideaki Sato, Masakazu Sugishima, Mai Tsukaguchi, Takahiro Masuko, Mikuru Iijima, Mitsunori Takano, Yoshiaki Omata, Kei Hirabayashi, Kei Wada, Yoshio Hisaeda, Ken Yamamoto

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)1023-1042
Number of pages20
JournalBiochemical Journal
Volume478
Issue number5
DOIs
Publication statusPublished - 2021 Mar

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Crystal structures of hydroxymethylbilane synthase complexed with a substrate analog: A single substrate-binding site for four consecutive condensation steps'. Together they form a unique fingerprint.

Cite this