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

Abstract

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 () 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.

Molecular Analysis of 55 Spanish Patients with Acute Intermittent Porphyria

“…Prokaryotic expression studies revealed that this change produces a protein with little, if any, residual activity. A previously identified disease-causing mutation at the same residue (p.K98R) had a similar activity when expressed in E coli [3,20].…”

“…The novel missense mutation p.K98N affects a residue that is invariant through evolution [2,3]. Lysine 98 is part of domain 1 of HMBS and interacts with the dipyrromethene cofactor at the active site [3]. Prokaryotic expression studies revealed that this change produces a protein with little, if any, residual activity.…”

“…AIP results from a partial deficiency of hydroxymethylbilane synthase (HMBS; EC 4.3.1.8), also named porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthetic pathway. Human HMBS is a cytoplasmic monomeric enzyme that catalyzes the polymerization of four porphobilinogen (PBG) molecules to form a linear tetrapyrrole (hydroxymethylbilane), using the dipyrromethene cofactor [2,3].…”

“…The housekeeping mRNA contains exon 1 joined to exons 3-15 with the translation start codon in exon 1, and encodes an enzyme that contains 361 amino acids (~42 KD), whereas the erythroid-specific mRNA contains exons 2-15 with the translation start site located within exon 3, and it encodes an enzyme of 344 amino acids (~40 KD) [10,11]. The crystal structure of the ubiquitous human HMBS reveals that it consists of three distinct α/β domains of a similar size, with the active site between domains 1 and 2 [2,3,12].…”

Acute Intermittent Porphyria: An Overview of Therapy Developments and Future Perspectives Focusing on Stabilisation of HMBS and Proteostasis Regulators

“…The pyrrole elongation ( Figure 3 ) is a stepwise condensation, which starts from a dipyrromethane (DPM) cofactor in the holoenzyme (E), creating enzyme-intermediate complexes bound to one, two or three PBG molecules, referred to as ES, ES 2 and ES 3 . In the last step, an enzyme-intermediate complex bound to four PBG molecules is generated, i.e., ES 4 , and quickly released from the cofactor by hydrolysis yielding the linear HMB, as reviewed by Bung et al [ 62 ]. The DPM cofactor, composed of two PBG molecules, is not incorporated in the product and remains covalently attached to the enzyme [ 63 , 64 ].…”