Physiological function of enzyme ALAS2

In vertebrate organisms, there are two genes encoding ALAS enzymes that belong to α-oxoamine synthase family of pyridoxalphosphate(PLP)-dependent enzymes^[1]. ALAS1 is a house-keeping gene expressed ubiquitously, in contrast ALAS2 (gene location Xp11.21) is specific for erythroid progenitor cells^[2]. Both catalyze initial step in biosynthesis of heme cofactor. While the heme cofactor associated with proteins is essential for several physiological processes, for example transport of oxygen in red blood cells, free heme is toxic and perturbations in its metabolic pathway resulting in accumulation of intermediates lead to various blood diseases^{[3] [4]}.

ALAS role in heme biosynthesis

The initial and final steps of 8-step heme biosynthetic pathway take place in the mitochondrial matrix. Since the ALAS mediate first reaction it is rate-limiting enzyme regulating the whole pathway, also known as a gatekeeper^[5]. It catalyzes PLP-dependent condensation of glycine and succinyl-CoA forming 5-aminolevulinic acid (ALA)^[6]. ALA is then transported to cytoplasm where it undergoes subsequent reactions and eventually moves back to the mitochondria to form heme^[7].

The underlying mechanism of the ALAS enzymatic reaction is induced-fit substrate binding via open-to-close conformational transition. At first, the glycine substrate binds to PLP, an active form of vitamin B6, creating an external aldimine. Following deprotonation of glycine enable nucleophilic attack on the second substrate succinyl-CoA. Consequent condensation and decarboxylation form the ALA product. The product release relies on regeneration of an internal aldimine between PLP and ALAS protein^[8].

Structure of human ALAS2

ALAS2 enzyme is an obligatory homodimer. The interface between two monomeric subunits contains two active sites. In the absence of the substrate each active site, specifically its catalytic lysine residue (Lys391), bounds one PLP molecule. There are three domains within one human ALAS2 monomer. That is N-terminal domain (Met1-Val142) which contains mitochondrial targeting sequence, conserved catalytic core (Phe143-Gly544) and C-terminal domain (Leu545-Ala587) which is specific for eukaryotes^{[9] [10]}.

The conserved catalytic core can be further divided into glycine-rich motif (His219-Ile229) and active site loop (Tyr500-Arg517)^[11]. This loop plays a critical role in regulation of product release, since it interacts with the autoinhibitory C-terminal domain forming a regulatory gate . The regulation of the enzyme is further imposed by a particular alpha-helix (Ser568-Phe575) which residues Glu569 and Glu571 form salt bridge network with Asp159 and Arg511 (Figure 1C-D). The network establishes the closed state preventing the transition to the open state, in other words, it blocks the PLP-bounded active site^[12].

Mutations causing blood diseases

Several types of ALAS2 gene mutations can lead to X-linked blood disorders such as sideroblastic anemia and protoporphyria. These types of disease-causing mutations include missense, deletions, frameshifts.

X-linked sideroblastic anemia

XLSA is the most common form of sideroblastic anemias, because of ALAS2 localization on the X chromosome, the disease is more common in males^[13]. It is caused by various mutations throughout the sequence of ALAS2 enzyme. So far, 91 mutations have been described, mainly in the catalytic core but also in the C-terminal domain^[14]. These diverse mutations result in a common phenotype of reduced heme production and iron overload in erythroblasts. The effect of a mutation on ALAS2 varies from protein folding destabilization (e.g., Leu313Pro, Ile324Thr, Gly398Asp), loss-of-function aka decrease in enzymatic activity (e.g., Glu242Lys, Asp263Asn, Pro339Leu, Arg411His), interference with PLP binding site (e.g., Arg170His, Phe259Cys, Asp357Val) to changes in protein-protein interaction (e.g., Met567Val, Ser568Gly)^{[15] [16] [17]}.

This disease belongs to the group of hemoglobinopathies. It is characterized by microcytic hypochromic anemia and hypochromic anemia with the presence of iron-containing mitochondria surrounding the cell nucleus. These cells are called ring sideroblasts (erythrocyte precursors) and are found in the bone marrow of the patients^{[18] [19]}. The patients suffer from mild symptoms such as pallor, fatigue, dizziness, weight loss, heart rate acceleration and more fatal one as cardiac disease and cirrhosis. As a result of secondary toxicity of accumulated iron, diabetes may develop citace. Differential diagnosis requires detection of ring sideroblasts in the bone marrow by iron staining. Next-generation genome sequencing is required to rule out reversible causes^[20]. Typical treatment for patients with XLSA is pyridoxine supplementation. However, some patients with mutations in PLP-binding site (e.g., Asp357Val) do not respond to pyridoxine treatment. On the other hand, most patients that are responsive to the treatment does not carry PLP-binding site mutations^[21].

X-linked protoporphyria

X-linked protoporphyria is a rare genetic disorder that belongs to the group of photodermatoses^[22]. It results from a mutation in gene sequence for the C-terminal domain of ALAS2. There are several types of mutations including deletion, missense and frameshift, which cause the protein to be truncated or elongated compared to wild type. One of these mutations (ΔG) is associated with increased stability of the enzyme, while the others cause hyperactivity of the enzyme^{[23] [24] [25]}. Deletions result in truncation or frameshift of this autoinhibitory domain disrupt molecular interactions that maintain strict regulation of enzyme activity. The consequence is lower inhibition and thus the aforementioned hyperactivity of the enzyme. Due to the higher activity of ALAS2, toxic heme intermediates accumulate in erythrocytes.

The most common symptom of XLP is phototoxicity within minutes after exposure to direct sunlight. It is characterized by burning, itching, tingling, pain and redness of the skin, and blisters may appear rarely, accompanied with swelling and scarring when prolonged sun exposition. Repeated episodes of phototoxicity can lead to permanent and chronic skin changes. Some patients can develop severe symptoms such as enlargement of the spleen and chronic kidney disease. A defect in the heme biosynthetic pathway in those affected leads to the accumulation of protoporphyrin in erythrocytes, which is subsequently released into the plasma and uptaken by the liver and vascular endothelium. The accumulated protoporphyrin becomes activated upon exposure to sunlight and begins to produce singlet oxygen radical reactions that result in tissue damage. Some patients may also develop hepatic dysfunction leading to liver failure due to the deposition of protoporphyrin in bile or hepatocytes. In association with liver failure, some patients may also develop motor neuropathy. In addition, excess protoporphyrin is also linked to the formation of gallstones. Patients with XLP also often suffer from vitamin D deficiency due to sun avoidance^{[26] [27]}.

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