Human aminolevulinate synthase structure reveals a eukaryotic-specific autoinhibitory loop regulating substrate binding and product release

Abstract

Abstract5′-aminolevulinate synthase (ALAS) catalyzes the first step in heme biosynthesis, generating 5′-aminolevulinate from glycine and succinyl-CoA. Inherited frameshift indel mutations of human erythroid-specific isozyme ALAS2, within a C-terminal (Ct) extension of its catalytic core that is only present in higher eukaryotes, lead to gain-of-function X-linked protoporphyria (XLP). Here, we report the human ALAS2 crystal structure, revealing that its Ct-extension folds onto the catalytic core, sits atop the active site, and precludes binding of substrate succinyl-CoA. The Ct-extension is therefore an autoinhibitory element that must re-orient during catalysis, as supported by molecular dynamics simulations. Our data explain how Ct deletions in XLP alleviate autoinhibition and increase enzyme activity. Crystallography-based fragment screening reveals a binding hotspot around the Ct-extension, where fragments interfere with the Ct conformational dynamics and inhibit ALAS2 activity. These fragments represent a starting point to develop ALAS2 inhibitors as substrate reduction therapy for porphyria disorders that accumulate toxic heme intermediates.

A hemizygous p.R204Q mutation in the ALAS2 gene underlies X-linked sideroblastic anemia in an adult Chinese Han man

“…We detected a missense mutation for a G to A transition in exon 5 of the ALAS2 gene, which caused a arginine (Arg) to glutamine (Gln) substitution at amino acid position 204 (NM_000032.5: c.611G>A; p.R204Q) in the proband and his daughter (Fig. 2 a, c), which located at the domains important for pyridoxal phosphate co-factor binding [ 6 , 7 ]. His son (III-1) was normal (Fig.…”

“…In the process of ALA synthesis, PLP is a cofactor reversibly bound to Lysine 391 and its binding is absolutely required for enzymatic activity [ 15 ]. All reported ALAS2 mutations in male XLSA patients were missense mutations, mostly located at the domains important for pyridoxal phosphate co-factor binding or catalysis [ 6 , 7 ]. Pyridoxine can enhance the activity of ALAS2 enzyme, more than half of XLSA patients have response to pyridoxine [ 1 ].…”

“…Pyridoxine can enhance the activity of ALAS2 enzyme, more than half of XLSA patients have response to pyridoxine [ 1 ]. Our reported ALAS2 mutation (p.Arg204Gln) is located on the surface of the ALAS2 3D structure some distance from the PLP moiety that is buried in the middle of the structure [ 6 ]. We can only speculate that the presence of Gln at the R204 location destabilizes the enzyme's structure but the binding of the PLP cofactor stabilizes the mutant structure somewhat.…”

Cytochromes of Mitochondries and Activity of Heme Metabolism Enzymes in the Liver Under Different Nutrient Regimes

“…Оскільки цитохроми є гемовмісними протеїнами, то їх кількість значною мірою визначається збалансованістю процесів їх синтезу та катаболізму [9]. Ключовим ензимом метаболічного шляху синтезу гему є δ-амінолевулінатсинтаза (КФ 2.3.1.37), що каталізує першу реакцію біосинтетичного шляху гему -конденсацію гліцину та сукциніл-коензиму А з утворенням δ-амінолевулінової кислоти [10]. Гемоксигеназа (КФ 1.14.99.3)ключовий ензим деградації гему -забезпечує окисне розщеплення гему з утворенням в еквімолярній кількості монооксиду карбону (СО) та білівердину, що розглядаються як цитопротекторні молекули [11,12].…”

A Novel ALAS2 Missense Mutation in Two Brothers With Iron Overload and Associated Alterations in Serum Hepcidin/Erythroferrone Levels

“…The structural analysis of the missense variants was made based on the available human ALAS2 crystallographic structure (Bailey et al, 2020) (PDB accession code: 5QQQ, crystallographic resolution: 1.93 Å).…”