Structural studies of substrate and product complexes of 5-aminolaevulinic acid dehydratase from humans,Escherichia coliand the hyperthermophilePyrobaculum calidifontis

Abstract

A number of X-ray analyses of an enzyme involved in a key early stage of tetrapyrrole biosynthesis are reported. Two structures of human 5-aminolaevulinate dehydratase (ALAD), native and recombinant, have been determined at 2.8 Å resolution, showing that the enzyme adopts an octameric quaternary structure in accord with previously published analyses of the enzyme from a range of other species. However, this is in contrast to the finding that a disease-related F12L mutant of the human enzyme uniquely forms hexamers [Breinig et al. (2003), Nature Struct. Biol. 10, 757-763]. Monomers of all ALADs adopt the TIM-barrel fold; the subunit conformation that assembles into the octamer includes the N-terminal tail of one monomer curled around the (α/β) barrel of a neighbouring monomer. Both crystal forms of the human enzyme possess two monomers per asymmetric unit, termed A and B. In the native enzyme there are a number of distinct structural differences between the A and B monomers, with the latter exhibiting greater disorder in a number of loop regions and in the active site. In contrast, the second monomer of the recombinant enzyme appears to be better defined and the active site of both monomers clearly possesses a zinc ion which is bound by three conserved cysteine residues. In native human ALAD, the A monomer also has a ligand resembling the substrate ALA which is covalently bound by a Schiff base to one of the active-site lysines (Lys252) and is held in place by an ordered active-site loop. In contrast, these features of the active-site structure are disordered or absent in the B subunit of the native human enzyme. The octameric structure of the zinc-dependent ALAD from the hyperthermophile Pyrobaculum calidifontis is also reported at a somewhat lower resolution of 3.5 Å. Finally, the details are presented of a high-resolution structure of the Escherichia coli ALAD enzyme co-crystallized with a noncovalently bound moiety of the product, porphobilinogen (PBG). This structure reveals that the pyrrole side-chain amino group is datively bound to the active-site zinc ion and that the PBG carboxylates interact with the enzyme via hydrogen bonds and salt bridges with invariant residues. A number of hydrogen-bond interactions that were previously observed in the structure of yeast ALAD with a cyclic intermediate resembling the product PBG appear to be weaker in the new structure, suggesting that these interactions are only optimal in the transition state.

GATA/Heme Multi-omics Reveals a Trace Metal-Dependent Cellular Differentiation Mechanism

“…As the multi-omic analysis revealed essential hemoglobin components and the critical regulator of late-stage erythroid maturation UBE2O (Nguyen et al, 2017), a ubiquitin-conjugating enzyme, we considered whether proteins regulated similarly also have vital roles in erythrocyte biology. The zinc finger transcription factors GATA-1 (Evans and Felsenfeld, 1989; Pevny et al, 1991; Tsai et al, 1989) and KLF1 (Miller and Bieker, 1993; Perkins et al, 1995) control erythrocyte differentiation, and zinc is a critical cofactor of the heme biosynthetic enzyme delta-aminolevulinic acid dehydratase (ALAD) (Mills-Davies et al, 2017). How these factors sense changes in environmental and intracellular zinc and whether alterations in intracellular zinc impact the activity of these proteins are unclear.…”

“…Zinc increases activity of the zinc-finger transcription factor Metal Transcription Factor 1 (MTF-1), which activates transcription of zinc-regulatory genes, including metallothioneins, intracellular zinc-chelating proteins (Gunther et al, 2012). The octameric ALAD heme biosynthesis enzyme contains eight zinc atoms required for enzymatic activity (Mills-Davies et al, 2017). While iron insertion into protoporphyrin IX forms heme physiologically, in iron deficiency, zinc replaces iron to yield zinc protoporphyrin IX.…”

Dependence of heme biosynthesis on iron-sulfur cluster biogenesis: human ALAD is an unrecognized iron-sulfur protein

“…Consistent with these ndings, a Cys132Arg mutation was found to be responsible for ALAD porphyria in a 14-year-old boy 61 . Furthermore, we speculate that the structure of ALAD with a [Fe 4 S 4 ] cluster may resemble the crystal structure of Zn-ALAD ( Figure S6) 39 , and Cys residues 122, 124, and 132, the Zn(II) ligands in the published structures of ALAD 39 , might coordinate the [Fe 4 S 4 ] cofactor, of which the unique, non-Cyscoordinated Fe site can be coordinated by the ALA substrate 54,57,62 . Cys119 and Cys162 are located on two parallel beta-strands ( Figure S6) in positions where they have been proposed to form a disul de bond that stabilizes ALAD tertiary structure 38 .…”

“…[Fe 4 S 4 ]-ALAD has signi cantly higher enzymatic activity than zinc-ALAD ALAD was previously identi ed as a zinc enzyme, and structures with zinc in the active site have been studied 38,39 . Therefore, to determine whether the [Fe 4 S 4 ] cluster of human ALAD is functionally signi cant, we compared the enzymatic activities of aerobically puri ed wild-type apo-ALAD (aero-apo-ALAD WT, see the Methods section for details), aerobically puri ed wild-type ALAD (aero-ALAD WT, aerobically puri ed wild-type ALAD prior to treating with EDTA, ), aerobically puri ed wild-type zinc bound-ALAD (aero-Zn-ALAD WT), and the anaerobically puri ed wild-type ALAD.…”

In Silico Studies of Mammalian δ‐ALAD Interactions with Selenides and Selenoxides

“…The 3D structure of human δ‐ALAD was obtained from the Protein Data Bank (http://www.rcsb.org/pdb/) with the code: 5HMS . The Chimera 1.8 software was used to remove the chain B, water and other molecules, and to add hydrogen atoms to the protein.…”