Human hydroxymethylbilane synthase: Molecular dynamics of the pyrrole chain elongation identifies step-specific residues that cause AIP

Abstract

<p id="d4631774e272">Human hydroxymethylbilane synthase (hHMBS) is a monomeric enzyme that catalyzes the stepwise head-to-tail condensation of four porphobilinogen (PBG) molecules to form the linear tetrapyrrole 1-hydroxymethylbilane (HMB). Mutations in the <i>hHMBS</i> gene cause autosomal-dominant acute intermittent porphyria (AIP). Although crystal structures of hHMBS have been reported, the specific active-site residues and the molecular mechanism of the stepwise PBG chain elongation are unknown. Here, by using molecular-dynamics simulations, the mechanisms and active-site residues for the HMB stepwise synthesis and HMB exit were determined. Mutagenesis of key active-site residues and in vitro expression studies identified the molecular basis of mutations causing AIP. </p><p class="first" id="d4631774e278">Hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway, catalyzes the head-to-tail condensation of four molecules of porphobilinogen (PBG) to form the linear tetrapyrrole 1-hydroxymethylbilane (HMB). Mutations in human <i>HMBS</i> ( <i>hHMBS</i>) cause acute intermittent porphyria (AIP), an autosomal-dominant disorder characterized by life-threatening neurovisceral attacks. Although the 3D structure of hHMBS has been reported, the mechanism of the stepwise polymerization of four PBG molecules to form HMB remains unknown. Moreover, the specific roles of each of the critical active-site residues in the stepwise enzymatic mechanism and the dynamic behavior of hHMBS during catalysis have not been investigated. Here, we report atomistic studies of HMB stepwise synthesis by using molecular dynamics (MD) simulations, mutagenesis, and in vitro expression analyses. These studies revealed that the hHMBS active-site loop movement and cofactor turn created space for the elongating pyrrole chain. Twenty-seven residues around the active site and water molecules interacted to stabilize the large, negatively charged, elongating polypyrrole. Mutagenesis of these active-site residues altered the binding site, hindered cofactor binding, decreased catalysis, impaired ligand exit, and/or destabilized the enzyme. Based on intermediate stages of chain elongation, R26 and R167 were the strongest candidates for proton transfer to deaminate the incoming PBG molecules. Unbiased random acceleration MD simulations identified R167 as a gatekeeper and facilitator of HMB egress through the space between the enzyme’s domains and the active-site loop. These studies identified the specific active-site residues involved in each step of pyrrole elongation, thereby providing the molecular bases of the active-site mutations causing AIP. </p>