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

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 Šresolution, 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.

Coupling of Redox and Structural States in Cytochrome P450 Reductase Studied by Molecular Dynamics Simulation.

Cytochrome P450 reductase (CPR) is the key protein that regulates the electron transfer from NADPH to various heme-containing monooxygenases. CPR has two flavin-containing domains: one with flavin adenine dinucleotide (FAD), called FAD domain, and the other with flavin mononucleotide (FMN), called FMN domain. It is considered that the electron transfer occurs via FAD and FMN (NADPH → FAD → FMN → monooxygenase) and is regulated by an interdomain open-close motion. It is generally thought that the structural state is coupled with the redox state, which, however, has not yet been firmly established. In this report, we studied the coupling of the redox and the structural states by full-scale molecular dynamics (MD) simulation of CPR (total 86.4 µs). Our MD result showed that while CPR predominantly adopts the closed state both in the oxidized and reduced states, it exhibits a tendency to open in the reduced state. We also found a correlation between the FAD-FMN distance and the predicted FMN-monooxygenase distance, which is embedded in the equilibrium thermal fluctuation of CPR. Based on these results, a physical mechanism for the electron transfer by CPR is discussed.