Structure of the Mitochondrial Aminolevulinic Acid Synthase, a Key Heme Biosynthetic Enzyme

Abstract

5-Aminolevulinic acid synthase (ALAS) catalyzes the first step in heme biosynthesis. We present the crystal structure of a eukaryotic ALAS from Saccharomyces cerevisiae. In this homodimeric structure, one ALAS subunit contains covalently bound cofactor, pyridoxal 5'-phosphate (PLP), whereas the second is PLP free. Comparison between the subunits reveals PLP-coupled reordering of the active site and of additional regions to achieve the active conformation of the enzyme. The eukaryotic C-terminal extension, a region altered in multiple human disease alleles, wraps around the dimer and contacts active-site-proximal residues. Mutational analysis demonstrates that this C-terminal region that engages the active site is important for ALAS activity. Our discovery of structural elements that change conformation upon PLP binding and of direct contact between the C-terminal extension and the active site thus provides a structural basis for investigation of disruptions in the first step of heme biosynthesis and resulting human disorders.

Keywords: AAA+ unfoldase; ClpX; XLPP; XLSA; porphyria; sideroblastic anemia; α-oxoamine family.

Figure 1. Structure of ALAS from S. cerevisiae

A. Cartoon representation of the ALAS dimer with subunits A (unliganded) and B (containing covalently bound lysine-PLP (Lys-PLP)) colored tan and green, respectively. Lys–PLP is shown as blue spheres. The unoccupied cofactor-binding pocket is outlined with a blue dotted line. The structured N- and C-termini from Subunit A are labeled with name and residue number. B. PLP binding pocket containing the covalently bound cofactor with 2mFo-Fc electron density for Lys–PLP ligand contoured at 1.0 σ and shown as grey mesh. Residues that directly interact with PLP are shown as sticks and are labeled according to their subunit with a superscript notation. See also Figure S1.

Figure 2. ALAS structure reveals regions that become ordered by PLP

A. Overlay of ALAS_Sc subunits colored as in Fig. 1A, with emphasis on the PLP-ordered regions. NT (residues 83–113) is colored magenta. GR (residues 147–156) is colored orange. CT (residues 538–548) is colored cyan. All PLP-responsive regions are represented in three of the six molecules in the crystallographic asymmetric unit, however the lengths of NT and CT vary slightly depending on the molecule (NT in Chain D is residues 82–113 and CT in Chain C is residues 535–548). The segments reported here represent the shortest disordered stretch of residues among all comparable molecules. B. Asymmetric ALAS_Sc homodimer (top panel is the same orientation as Figure 1A, Lys–PLP shown as blue spheres) with the NT, GR, and CT regions depicted at approximately 2X magnification, and colored as in panel A. C. Overlay of PLP-binding pockets from both subunits. The major conformational changes between the unbound and bound pockets are shown as black arrows, with the arrowhead marking the position in the bound pocket. See also Figure S1B.

Figure 3. Model of ALAS_Sc active sites with cofactor and substrates

A. ALAS dimer colored as in Figure 1 containing bound Lys–PLP (blue spheres) with glycine-PLP (yellow spheres) and succinyl-CoA (purple spheres) modeled in. Substrates were modeled based upon superposition of ALAS_Sc onto ALAS_Rc structures (PDB 2BWP and 2BWO). B. Close-up view of the PLP-containing ALAS_Sc active site with modeled substrates. Residues known to interact with substrates based on alignment with ALAS_Rc are shown as sticks. Residues that become ordered by PLP binding (Arg91^B, Thr150^A, Asn152^A, Ile153^A) or move after PLP binding (His209^B and Phe365^A) are labeled with red font. C. Close-up view of the PLP-free ALAS_Sc active site with modeled substrates. Residues Arg91^A, Thr150^B, Asn152^B, and Ile153^B are disordered with respect to this active site. See also Figure S2.

Figure 4. PLP stabilizes regions required for substrate binding

A. Interactions between NT (magenta), GR (orange), and the active-site loop (brown). Thr452^B, a conserved residue in the active-site loop, interacts with Met257^B, Ile448^B, and Arg91^B in NT. GR packs against part of NT (see Figure S3A). B. Interactions between GR (orange) and CT (cyan) stabilized by PLP. Hydrogen-bonding network between Arg151^A from GR, Glu164^A from the core, and the backbone carbonyl of Ser543^A in CT (cyan) depicted as black dotted lines. ALAS_Sc pyridoxyl-lysine (blue), ALAS_Rc pyridoxyl-glycine (yellow), and ALAS_Rc succinyl-CoA (purple) are shown as spheres. All of the colored regions are disordered in the absence of PLP, including Thr150, Asn152 and Ile153, which directly contribute to succinyl-CoA binding. See also Figure S2C,E.

Figure 5. Structure and function of the ALAS eukaryote-specific C-terminal extension

A. The asymmetric ALAS_Sc dimer is shown as a surface representation with subunits colored tan or green. The eukaryote-specific C-terminal extension (residues 489–548, cyan cartoon) wraps around the outer surface of the dimer and packs into a hydrophobic channel. B. In vitro activity of ALAS_Sc WT, R151A mutant, or ΔCT (residues 58–534) at 30 °C. Experiments were performed in triplicate and error bars represent SEM. C. Growth of yeast strains harboring C-terminal deletion mutants of ALAS_Sc. The indicated variants in HEM1 (the gene encoding ALAS_Sc) were integrated at its single genomic locus in W303a strain background (see Table S2). Five-fold serial dilutions from cell suspensions with OD₆₀₀ = 1 were spotted on YP + 2% agar, + 2% glucose or 3% glycerol, ± 50 µg/mL ALA as indicated, and grown for 2 d (+ glucose) or 3 d (+ glycerol) at 30 °C. D. Cellular protein levels of C-terminal ALAS_Sc variants. ALAS_Sc was detected by FLAG antibody (M2 clone, Sigma), and the mitochondrial outer-membrane porin Por1 was probed as a loading control. E. ALA levels in yeast cell extracts were measured using modified Ehrlich’s reagent (described in STAR methods) and normalized to wild type. Data are represented as mean ± SD; p ≤ 0.005 for ALA reduction in all HEM1 mutants (Student’s t-test, n = 3 (biological replicates)). See also Figure S4.