Elucidating the Role of Human ALAS2 C-terminal Mutations Resulting in Loss of Function and Disease

Abstract

The conserved enzyme aminolevulinic acid synthase (ALAS) initiates heme biosynthesis in certain bacteria and eukaryotes by catalyzing the condensation of glycine and succinyl-CoA to yield aminolevulinic acid. In humans, the ALAS isoform responsible for heme production during red blood cell development is the erythroid-specific ALAS2 isoform. Owing to its essential role in erythropoiesis, changes in human ALAS2 (hALAS2) function can lead to two different blood disorders. X-linked sideroblastic anemia results from loss of ALAS2 function, while X-linked protoporphyria results from gain of ALAS2 function. Interestingly, mutations in the ALAS2 C-terminal extension can be implicated in both diseases. Here, we investigate the molecular basis for enzyme dysfunction mediated by two previously reported C-terminal loss-of-function variants, hALAS2 V562A and M567I. We show that the mutations do not result in gross structural perturbations, but the enzyme stability for V562A is decreased. Additionally, we show that enzyme stability moderately increases with the addition of the pyridoxal 5'-phosphate (PLP) cofactor for both variants. The variants display differential binding to PLP and the individual substrates compared to wild-type hALAS2. Although hALAS2 V562A is a more active enzyme in vitro, it is less efficient concerning succinyl-CoA binding. In contrast, the M567I mutation significantly alters the cooperativity of substrate binding. In combination with previously reported cell-based studies, our work reveals the molecular basis by which hALAS2 C-terminal mutations negatively affect ALA production necessary for proper heme biosynthesis.

Figure 1

Experimental and computational hALAS2 models display C-terminal flexibility. (A–C) The catalytic core (residues 140-547) of human ALAS2 is shown as a surface representation with one protomer in tan and the symmetry-related protomer in gray. The C-terminal extension (residues 548-587) of each model is shown in cartoon representation and colored as indicated. The flexible N-terminal extensions (residues 1-139) for all AlphaFold models are not shown for clarity. (A) The overlay of the WT human ALAS2 homodimer from the crystal structure (PDB ID 6HRH) and AlphaFold predicted model. The C-terminal extensions are colored blue and pink, respectively. (B) Superposition of the hALAS2 crystal structure (blue) and the AlphaFold models of hALAS2 WT (pink) and hALAS2 V562A (teal). Residue 562 is shown as spheres. (C) Superposition of the hALAS2 crystal structure (blue) and the AlphaFold models of hALAS2 WT (pink) and hALAS2 M567I (purple). Residue 567 is shown as spheres. Complete AlphaFold models colored based on pLDDT score are shown in Figure S1.

Figure 2

Impact of C-terminal mutations on enzyme structure and stability. (A) Circular dichroism polarimetry analysis of WT, V562A, and M567I hALAS2 variants. (B, C) The unfolding temperatures of holo hALAS2 variants (B) or apo hALAS2 variants (C) assayed by differential scanning fluorimetry in the absence (solid bars) or presence (hatched bars) of exogenous PLP (****p < 0.0001, ns: not significant).

Figure 3

Absorbance and fluorescence spectroscopy reveal the mode of steady-state PLP binding. (A) UV–visible absorption spectra of hALAS2 WT (black), V562A (teal), and M567I (purple). The right panel is zoomed into the region between 300–500 nm. The substituted aldimine and enolimine tautomers absorb around 326 nm and the ketoenamine tautomer absorbs near 424 nm. (B) Fluorescence emission spectra for WT, V562A, and M567I hALAS2 proteins, colored as in panel (A), with excitation at 326 nm. The single peak at 380 nm is indicative of the inactive substituted aldimine species. (C) The ratio of the active ketoenamine population compared to the total PLP bound was determined by the area under the curve of the absorption spectra (**p = 0.0075, ****p < 0.0001).

Figure 4

Steady-state activity of hALAS2 variants in the absence and presence of exogenous PLP. Maximal enzyme activity for hALAS2 variants was determined under saturating substrate concentrations in the absence (solid bars) or presence (hatched bars) of exogenous PLP. The addition of excess PLP did not affect hALAS2 WT (gray), V562A (teal), or M567I (purple) activity (*p = 0.0477, ****p < 0.0001, ns: not significant).

Figure 5

PLP binding kinetics for hALAS2 variants. The activity of the hALAS2 apoenzyme preparations was determined as a function of increasing PLP concentration. Data were normalized by subtracting the signal in the presence of 0 nM PLP. Each experiment was performed with a minimum of three biological replicates, each containing three technical replicates (data points represent the technical replicates).

Figure 6

Substrate binding kinetics for hALAS2 variants. (A) The activity of the hALAS2 variants was determined as a function of varied glycine (A) or succinyl-CoA (B) concentrations. Data were fit with a Michaelis–Menten model for hALAS2 WT and V562A and with an allosteric sigmoidal model for hALAS2 M567I. Each experiment was performed with a minimum of three biological replicates, each containing three technical replicates (data points represent the technical replicates).

Figure 7

hALAS2 mutations alter enzyme activity via different mechanisms. Summary of the molecular impact of hALAS2 C-terminal mutations on enzyme structure and function. ALAS2 enzymes are depicted as either a large gray (WT), teal (V562A), or purple (M567I) oval. The substrates are represented in light blue (glycine) and green (succinyl-CoA) shapes and the PLP cofactor is represented as a yellow circle. The V562A variant displays decreased stability (blurred outline), abrogated succinyl-CoA binding affinity (hatched substrate rectangle), and catalytic efficiency. In contrast, the M567I variant binds both substrates with negative cooperativity (broken shapes) and has impaired enzyme activity, which may be the primary driver of hALAS2 loss of function underlying X-linked sideroblastic anemia.