ALAS2 in erythroid heme biosynthesis disorders

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ALAS2 in erythroid heme biosynthesis disorders

5’-aminolevulinic acid synthase (ALAS, EC 2.3.1.37) catalyzes the first step in biosynthesis of heme molecule in α-proteobacteria and mitochondria of nonplant eukaryotes. In vertebrates there are two isoforms of ALAS enzyme. The erythroid-specific ALAS2 located on chromosome X is expressed during erythropoiesis and mediates biosynthesis of heme that carries oxygen in hemoglobin. Different mutations thorough the sequence of the enzyme lead to two ALAS2-associated blood disorders. Namely X-linked sideroblastic anemia (XLSA, MIM 300751) and X-linked protoporphyria (XLP, MIM 300752) typically caused by loss-of function (enzyme deficiency) and gain-of-function (enzyme hyperactivity), respectively.

Physiological function of enzyme ALAS2

In vertebrates, 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 mitochondrial matrix. Since the ALAS mediate first reaction it is rate-limiting enzyme regulating the whole pathway, also known as the 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[3]. 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[7].

Structure of human ALAS2

Structure of human ALAS2 enzyme

Drag the structure with the mouse to rotate

ALAS2 enzyme is an obligatory . 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. They are N-terminal domain (Met1-Val142), which contains mitochondrial targeting sequence, (Phe143-Gly544) and (Leu545-Ala587) which is specific for eukaryotes[8][9]. The conserved catalytic core can be further divided into (His219-Ile229) and (Tyr500-Arg517). This loop plays a critical role in regulation of product release, since it interacts with the autoinhibitory C-terminal domain forming a regulatory gate[10]. The regulation of the enzyme is further imposed by a particular (Ser568-Phe575) whose residues Glu569 and Glu571 form salt bridge network with Asp159 and Arg511. The network establishes the closed state preventing the transition to the open state, in other words, it blocks the PLP-bounded active site[8].

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[11]. It is caused by various mutations throughout the sequence of ALAS2 enzyme. So far, 91 mutations have been described. They are mainly located in the catalytic core but can be also detected in the C-terminal domain[10]. 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 (e.g., Leu313Pro, Ile324Thr, Gly398Asp), loss-of-function aka decrease in (e.g., Glu242Lys, Asp263Asn, Pro339Leu, Arg411His), interference with (e.g., Arg170His, Phe259Cys, Asp357Val) to changes in (e.g., Met567Val, Ser568Gly)[12][13][14].

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[13][15]. The patients suffer from mild symptoms such as fatigue, dizziness, sensations ranging from weight loss, heart rate acceleration and more fatal one as cardiac disease and cirrhosis. Differential diagnosis requires detection of ring sideroblasts in the bone marrow by iron staining with the possibility of next-generation genome sequencing to exclude reversible causes[15]. 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 treatment do not carry PLP-binding site mutations[10].

X-linked protoporphyria

X-linked protoporphyria is a rare genetic disorder that belongs to the group of photodermatoses[16]. 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:

  • WT: LPLQDVSVAACNFCRRPVHFELMSEWERSYFGNMGPQYVTTYA (587 aa long ALAS2)
  • Q548X: LPL (548 aa long ALAS2)
  • ΔAT: LPLQDVSVAACNFCRRPVHFELE (567 aa long ALAS2)
  • ΔATGT: LPLQDVSVAACNFCRRPVHFELMSGNVPTSGTWGPSMSPPMPEKPAA (591 aa long ALAS2)
  • Δ26bp: LPLQDVPSCTL (556 aa long ALAS2)
  • ΔG: LPLQDVSVAACNFCRRPVHFELMSEWERSYFGNMGPSMSPPMPKEKPAA (592 aa long ALAS2)


One of these mutations (ΔG) is associated with increased stability of the enzyme, while the others cause hyperactivity of the enzyme[17][18]. Deletions 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 16 repeat [16][19]. Diagnosis is based on the determination of significantly elevated levels of free erythrocytic protoporphyrin and a predominance of metal-free protoporphyrin. The diagnosis is subsequently confirmed by sequencing of the ALAS2 gene. There is no specific therapy for X-linked protoporphyria yet. The therapy mainly focuses on suppressing the symptoms of the disease. The main focus of treatment for protoporphyria is prevention, which consists of limiting exposure to direct sunlight and ultraviolet light[16][19].


Adéla Fejfarová/Sandbox 3


References

  1. ↑ Eliot AC, Kirsch JF. Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations. Annu Rev Biochem. 2004;73:383-415. doi: 10.1146/annurev.biochem.73.011303.074021. PMID:15189147 doi:http://dx.doi.org/10.1146/annurev.biochem.73.011303.074021
  2. ↑ doi: https://dx.doi.org/10.1074/jbc.M111.3064232
  3. ↑ 3.0 3.1 Dailey HA, Meissner PN. Erythroid heme biosynthesis and its disorders. Cold Spring Harb Perspect Med. 2013 Apr 1;3(4):a011676. doi:, 10.1101/cshperspect.a011676. PMID:23471474 doi:http://dx.doi.org/10.1101/cshperspect.a011676
  4. ↑ Chiabrando D, Mercurio S, Tolosano E. Heme and erythropoieis: more than a structural role. Haematologica. 2014 Jun;99(6):973-83. doi: 10.3324/haematol.2013.091991. PMID:24881043 doi:http://dx.doi.org/10.3324/haematol.2013.091991
  5. ↑ Hunter GA, Ferreira GC. Molecular enzymology of 5-aminolevulinate synthase, the gatekeeper of heme biosynthesis. Biochim Biophys Acta. 2011 Nov;1814(11):1467-73. doi:, 10.1016/j.bbapap.2010.12.015. Epub 2011 Jan 6. PMID:21215825 doi:http://dx.doi.org/10.1016/j.bbapap.2010.12.015
  6. ↑ doi: https://dx.doi.org/10.1016/S0021-9258(19)77371-2
  7. ↑ Hunter GA, Zhang J, Ferreira GC. Transient kinetic studies support refinements to the chemical and kinetic mechanisms of aminolevulinate synthase. J Biol Chem. 2007 Aug 10;282(32):23025-35. doi: 10.1074/jbc.M609330200. Epub 2007, May 7. PMID:17485466 doi:http://dx.doi.org/10.1074/jbc.M609330200
  8. ↑ 8.0 8.1 Bailey HJ, Bezerra GA, Marcero JR, Padhi S, Foster WR, Rembeza E, Roy A, Bishop DF, Desnick RJ, Bulusu G, Dailey HA Jr, Yue WW. Human aminolevulinate synthase structure reveals a eukaryotic-specific autoinhibitory loop regulating substrate binding and product release. Nat Commun. 2020 Jun 4;11(1):2813. doi: 10.1038/s41467-020-16586-x. PMID:32499479 doi:http://dx.doi.org/10.1038/s41467-020-16586-x
  9. ↑ Kadirvel S, Furuyama K, Harigae H, Kaneko K, Tamai Y, Ishida Y, Shibahara S. The carboxyl-terminal region of erythroid-specific 5-aminolevulinate synthase acts as an intrinsic modifier for its catalytic activity and protein stability. Exp Hematol. 2012 Jun;40(6):477-86.e1. doi: 10.1016/j.exphem.2012.01.013. Epub, 2012 Jan 21. PMID:22269113 doi:http://dx.doi.org/10.1016/j.exphem.2012.01.013
  10. ↑ 10.0 10.1 10.2 Taylor JL, Brown BL. Structural basis for dysregulation of aminolevulinic acid synthase in human disease. J Biol Chem. 2022 Mar;298(3):101643. doi: 10.1016/j.jbc.2022.101643. Epub 2022, Jan 28. PMID:35093382 doi:http://dx.doi.org/10.1016/j.jbc.2022.101643
  11. ↑ Camaschella C. Recent advances in the understanding of inherited sideroblastic anaemia. Br J Haematol. 2008 Oct;143(1):27-38. doi: 10.1111/j.1365-2141.2008.07290.x. Epub, 2008 Jul 14. PMID:18637800 doi:http://dx.doi.org/10.1111/j.1365-2141.2008.07290.x
  12. ↑ Bishop DF, Tchaikovskii V, Hoffbrand AV, Fraser ME, Margolis S. X-linked sideroblastic anemia due to carboxyl-terminal ALAS2 mutations that cause loss of binding to the beta-subunit of succinyl-CoA synthetase (SUCLA2). J Biol Chem. 2012 Aug 17;287(34):28943-55. doi: 10.1074/jbc.M111.306423. Epub, 2012 Jun 27. PMID:22740690 doi:http://dx.doi.org/10.1074/jbc.M111.306423
  13. ↑ 13.0 13.1 Ducamp S, Schneider-Yin X, de Rooij F, Clayton J, Fratz EJ, Rudd A, Ostapowicz G, Varigos G, Lefebvre T, Deybach JC, Gouya L, Wilson P, Ferreira GC, Minder EI, Puy H. Molecular and functional analysis of the C-terminal region of human erythroid-specific 5-aminolevulinic synthase associated with X-linked dominant protoporphyria (XLDPP). Hum Mol Genet. 2013 Apr 1;22(7):1280-8. doi: 10.1093/hmg/dds531. Epub 2012 Dec, 20. PMID:23263862 doi:http://dx.doi.org/10.1093/hmg/dds531
  14. ↑ Liu G, Guo S, Kang H, Zhang F, Hu Y, Wang L, Li M, Ru Y, Camaschella C, Han B, Nie G. Mutation spectrum in Chinese patients affected by congenital sideroblastic anemia and a search for a genotype-phenotype relationship. Haematologica. 2013 Dec;98(12):e158-60. doi: 10.3324/haematol.2013.095513. PMID:24323989 doi:http://dx.doi.org/10.3324/haematol.2013.095513
  15. ↑ 15.0 15.1 Abu-Zeinah G, DeSancho MT. Understanding Sideroblastic Anemia: An Overview of Genetics, Epidemiology, Pathophysiology and Current Therapeutic Options. J Blood Med. 2020 Sep 25;11:305-318. doi: 10.2147/JBM.S232644. eCollection 2020. PMID:33061728 doi:http://dx.doi.org/10.2147/JBM.S232644
  16. ↑ 16.0 16.1 16.2 Balwani M. Erythropoietic Protoporphyria and X-Linked Protoporphyria: pathophysiology, genetics, clinical manifestations, and management. Mol Genet Metab. 2019 Nov;128(3):298-303. doi: 10.1016/j.ymgme.2019.01.020. Epub , 2019 Jan 24. PMID:30704898 doi:http://dx.doi.org/10.1016/j.ymgme.2019.01.020
  17. ↑ Ducamp S, Schneider-Yin X, de Rooij F, Clayton J, Fratz EJ, Rudd A, Ostapowicz G, Varigos G, Lefebvre T, Deybach JC, Gouya L, Wilson P, Ferreira GC, Minder EI, Puy H. Molecular and functional analysis of the C-terminal region of human erythroid-specific 5-aminolevulinic synthase associated with X-linked dominant protoporphyria (XLDPP). Hum Mol Genet. 2013 Apr 1;22(7):1280-8. doi: 10.1093/hmg/dds531. Epub 2012 Dec, 20. PMID:23263862 doi:http://dx.doi.org/10.1093/hmg/dds531
  18. ↑ Bishop DF, Tchaikovskii V, Nazarenko I, Desnick RJ. Molecular expression and characterization of erythroid-specific 5-aminolevulinate synthase gain-of-function mutations causing X-linked protoporphyria. Mol Med. 2013 Mar 5;19:18-25. doi: 10.2119/molmed.2013.00003. PMID:23348515 doi:http://dx.doi.org/10.2119/molmed.2013.00003
  19. ↑ 19.0 19.1 Balwani M, Desnick RJ. The porphyrias: advances in diagnosis and treatment. Blood. 2012 Nov 29;120(23):4496-504. doi: 10.1182/blood-2012-05-423186. Epub 2012, Jul 12. PMID:22791288 doi:http://dx.doi.org/10.1182/blood-2012-05-423186

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