RESUMO
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.
Assuntos
5-Aminolevulinato Sintetase , Anemia Sideroblástica , Humanos , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , 5-Aminolevulinato Sintetase/química , 5-Aminolevulinato Sintetase/deficiência , Anemia Sideroblástica/genética , Anemia Sideroblástica/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Mutação com Perda de Função , Estabilidade Enzimática , Heme/metabolismo , Heme/química , Porfirias/genética , Porfirias/metabolismo , Modelos Moleculares , Mutação , Protoporfiria EritropoéticaRESUMO
5-Aminolevulinic acid synthase (ALAS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of heme biosynthesis in α-proteobacteria and several non-plant eukaryotes. All ALAS homologs contain a highly conserved catalytic core, but eukaryotes also have a unique C-terminal extension that plays a role in enzyme regulation. Several mutations in this region are implicated in multiple blood disorders in humans. In Saccharomyces cerevisiae ALAS (Hem1), the C-terminal extension wraps around the homodimer core to contact conserved ALAS motifs proximal to the opposite active site. To determine the importance of these Hem1 C-terminal interactions, we determined the crystal structure of S. cerevisiae Hem1 lacking the terminal 14 amino acids (Hem1 ΔCT). With truncation of the C-terminal extension, we show structurally and biochemically that multiple catalytic motifs become flexible, including an antiparallel ß-sheet important to Fold-Type I PLP-dependent enzymes. The changes in protein conformation result in an altered cofactor microenvironment, decreased enzyme activity and catalytic efficiency, and ablation of subunit cooperativity. These findings suggest that the eukaryotic ALAS C-terminus has a homolog-specific role in mediating heme biosynthesis, indicating a mechanism for autoregulation that can be exploited to allosterically modulate heme biosynthesis in different organisms.
Assuntos
5-Aminolevulinato Sintetase , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , 5-Aminolevulinato Sintetase/química , Fosfato de Piridoxal/química , Domínio Catalítico , Heme/químicaRESUMO
The antibiotic alaremycin has a structure that resembles that of 5-aminolevulinic acid (ALA), a universal precursor of porphyrins, and inhibits porphyrin biosynthesis. Genome sequencing of the alaremycin-producing bacterial strain and enzymatic analysis revealed that the first step of alaremcyin biosynthesis is catalysed by the enzyme, AlmA, which exhibits a high degree of similarity to 5-aminolevulinate synthase (ALAS) expressed by animals, protozoa, fungi, and α-proteobacteria. Site-directed mutagenesis of AlmA revealed that the substitution of two amino acids residues around the substrate binding pocket transformed its substrate specificity from that of alaremycin precursor synthesis to ALA synthesis. To estimate the evolutionary trajectory of AlmA and ALAS, we performed an ancestral sequence reconstitution analysis based on a phylogenetic tree of AlmA and ALAS. The reconstructed common ancestral enzyme of AlmA and ALAS exhibited alaremycin precursor synthetic activity, rather than ALA synthetic activity. These results suggest that ALAS evolved from an AlmA-like enzyme. We propose a new evolutionary hypothesis in which a non-essential secondary metabolic enzyme acts as an 'evolutionary seed' to generate an essential primary metabolic enzyme.
Assuntos
5-Aminolevulinato Sintetase , 5-Aminolevulinato Sintetase/química , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Animais , Catálise , Mutagênese Sítio-Dirigida , Filogenia , Especificidade por SubstratoRESUMO
5-ALA is the precursor of all tetrapyrroles. 5-Aminolevulinate synthase (ALAS) catalyzes the production of 5-aminolevulinic acid (5-ALA) from glycine and succinyl-CoA. HemA from Rhodopseudomonas palustris (Rp-HemA) was reported to be a highly active ALAS. To understand the catalytic mechanism of Rp-HemA, the 2.05 Å resolution crystal structure of Rp-HemA was solved. Open, half close and close conformations were observed in the substrate-free structures. Structure comparison and sequence alignment suggest the newly observed half close conformation may also be conserved in ALAS family. The pre-existed close and half close conformations in Rp-HemA may play a key role for its high activity.
Assuntos
5-Aminolevulinato Sintetase , Rodopseudomonas , 5-Aminolevulinato Sintetase/química , Ácido Aminolevulínico , GlicinaRESUMO
Heme is a critical biomolecule that is synthesized in vivo by several organisms such as plants, animals, and bacteria. Reflecting the importance of this molecule, defects in heme biosynthesis underlie several blood disorders in humans. Aminolevulinic acid synthase (ALAS) initiates heme biosynthesis in α-proteobacteria and nonplant eukaryotes. Debilitating and painful diseases such as X-linked sideroblastic anemia and X-linked protoporphyria can result from one of more than 91 genetic mutations in the human erythroid-specific enzyme ALAS2. This review will focus on recent structure-based insights into human ALAS2 function in health and how it dysfunctions in disease. We will also discuss how certain genetic mutations potentially result in disease-causing structural perturbations. Furthermore, we use thermodynamic and structural information to hypothesize how the mutations affect the human ALAS2 structure and categorize some of the unique human ALAS2 mutations that do not respond to typical treatments, that have paradoxical in vitro activity, or that are highly intolerable to changes. Finally, we will examine where future structure-based insights into the family of ALA synthases are needed to develop additional enzyme therapeutics.
Assuntos
5-Aminolevulinato Sintetase , Anemia Sideroblástica , Doenças Genéticas Ligadas ao Cromossomo X , 5-Aminolevulinato Sintetase/química , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Ácido Aminolevulínico/química , Ácido Aminolevulínico/metabolismo , Anemia Sideroblástica/enzimologia , Anemia Sideroblástica/genética , Animais , Heme , Humanos , Relação Estrutura-AtividadeAssuntos
5-Aminolevulinato Sintetase/genética , Predisposição Genética para Doença , Proteína da Hemocromatose/genética , Sobrecarga de Ferro/diagnóstico , Sobrecarga de Ferro/etiologia , Mutação , 5-Aminolevulinato Sintetase/química , Adulto , Biomarcadores , Biópsia , Feminino , Estudos de Associação Genética , Proteína da Hemocromatose/química , Humanos , Perda de Heterozigosidade , Imageamento por Ressonância Magnética , Linhagem , Fenótipo , Índice de Gravidade de Doença , Relação Estrutura-Atividade , Avaliação de SintomasRESUMO
The caseinolytic mitochondrial matrix peptidase chaperone subunit (ClpX) plays an important role in the heme-dependent regulation of 5-aminolevulinate synthase (ALAS1), a key enzyme in heme biosynthesis. However, the mechanisms underlying the role of ClpX in this process remain unclear. In this in vitro study, we confirmed the direct binding between ALAS1 and ClpX in a heme-dependent manner. The substitution of C108 P109 [CP motif 3 (CP3)] with A108 A109 in ALAS1 resulted in a loss of ability to bind ClpX. Computational disorder analyses revealed that CP3 was located in a potential intrinsically disordered protein region (IDPR). Thus, we propose that conditional disorder-to-order transitions in the IDPRs of ALAS1 may represent key mechanisms underlying the heme-dependent recognition of ALAS1 by ClpX.
Assuntos
5-Aminolevulinato Sintetase/metabolismo , Endopeptidase Clp/metabolismo , Heme/metabolismo , Mitocôndrias/metabolismo , Chaperonas Moleculares/metabolismo , 5-Aminolevulinato Sintetase/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Hemina/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Biológicos , Ligação ProteicaRESUMO
5'-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.
Assuntos
5-Aminolevulinato Sintetase/química , Regulação Enzimológica da Expressão Gênica , 5-Aminolevulinato Sintetase/deficiência , 5-Aminolevulinato Sintetase/genética , Acil Coenzima A/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Doenças Genéticas Ligadas ao Cromossomo X/genética , Heme/química , Humanos , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Protoporfiria Eritropoética/genética , Especificidade por SubstratoAssuntos
5-Aminolevulinato Sintetase/genética , Anemia Sideroblástica/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação de Sentido Incorreto , Mutação Puntual , 5-Aminolevulinato Sintetase/química , Adulto , Anemia Sideroblástica/complicações , Medula Óssea/patologia , Sobrevivência Celular , Eritroblastos/patologia , Éxons/genética , Feminino , Genes Ligados ao Cromossomo X/genética , Heterozigoto , Humanos , Sobrecarga de Ferro/diagnóstico por imagem , Sobrecarga de Ferro/etiologia , Imageamento por Ressonância Magnética , Modelos Moleculares , Conformação Proteica , Sequenciamento do ExomaRESUMO
Iron-responsive elements (IREs) are ~35-nucleotide (nt) stem-loop RNA structures located in 5' or 3' untranslated regions (UTRs) of mRNAs that mediate post-transcriptional regulation by their association with IRE-binding proteins (IRPs). IREs are characterized by their apical 6-nt loop motif 5'-CAGWGH-3' (Wâ¯=â¯A or U and Hâ¯=â¯A, C or U), the so-called pseudotriloop, of which the loop nts C1 and G5 are paired, and the none-paired C between the two stem regions. In this study, the yeast three-hybrid (Y3H) system was used to investigate the relevance of the pseudotriloop structure of ferritin light chain (FTL) for the IRE-IRP interaction and the binding affinities between variant IRE(-like) structures and the two IRP isoforms, IRP1 and 2. Destabilization of the pseudotriloop structure by a G5-to-A mutation reduced binding of IRP1 and 2, while restoring the pseudotriloop conformation by the compensatory C1-to-U mutation, restored binding to both IRPs. In particular, IRP1 showed even stronger binding to the C1U-G5A mutant than to the wildtype FTL IRE. On the other hand, deletion of the bulged-out U6 of the pseudotriloop did not significantly affect its binding to either IRP1 or 2, but substitution with C particularly enhanced the binding to IRP1. In comparison to FTL IRE, IRE-like structures of 5'-aminolevulinate synthase 2 (ALAS2) and SLC40A1 (also known as ferroportin-1) showed similar or, in the case of endothelial PAS domain protein 1 (EPAS1) IRE, slightly weaker binding affinity to IRPs. SLC11A2 (a.k.a. divalent metal transporter-1) IRE exhibited relatively weak binding to IRP1 and medium binding to IRP2. Notably, the IRE-like structure of α-synuclein showed no detectable binding to either IRP under the conditions used in this Y3H assay. Our results indicate that Y3H can be used to characterize binding between IRPs and various IRE-like structures in vivo.
Assuntos
Apoferritinas/química , Apoferritinas/genética , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/metabolismo , 5-Aminolevulinato Sintetase/química , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Animais , Apoferritinas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Proteína 1 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/genética , Mutação , Conformação de Ácido Nucleico , Técnicas do Sistema de Duplo-Híbrido , Regiões não TraduzidasRESUMO
Identifying the viability of protein targets is one of the preliminary steps of drug discovery. Determining the ability of a protein to bind drugs in order to modulate its function, termed the druggability, requires a non-trivial amount of time and resources. Inability to properly measure druggability has accounted for a significant portion of failures in drug discovery. This problem is only further exacerbated by the large sample space of proteins involved in human diseases. With these barriers, the druggability space within the human proteome remains unexplored and has made it difficult to develop drugs for numerous diseases. Hence, we present a new feature developed in eFindSite that employs supervised machine learning to predict the druggability of a given protein. Benchmarking calculations against the Non-Redundant data set of Druggable and Less Druggable binding sites demonstrate that an AUC for druggability prediction with eFindSite is as high as 0.88. With eFindSite, we elucidated the human druggability space to be 10,191 proteins. Considering the disease space from the Open Targets Platform and excluding already known targets from the predicted data set reveal 2731 potentially novel therapeutic targets. eFindSite is freely available as a stand-alone software at https://github.com/michal-brylinski/efindsite .
Assuntos
Descoberta de Drogas/métodos , Proteínas/metabolismo , Aprendizado de Máquina Supervisionado , 5-Aminolevulinato Sintetase/química , 5-Aminolevulinato Sintetase/metabolismo , Sítios de Ligação , Desenho de Fármacos , Humanos , Ligação Proteica , Proteínas/química , Proteoma/química , Proteoma/metabolismo , Serina Proteases/química , Serina Proteases/metabolismo , SoftwareRESUMO
Strains of the phototrophic alpha-proteobacterium Rhodobacter sphaeroides vary in the number of enzymes catalyzing the formation of 5-aminolevulinic acid (ALA synthases) that are encoded in their genomes. All have hemA, but not all have hemT. This study compared transcription of these genes, and also properties of their products among three wild-type strains; 2.4.3 has hemA alone, 2.4.1 and 2.4.9 have both hemA and hemT. Using lacZ reporter plasmids all hemA genes were found to be upregulated under anaerobic conditions, but induction amplitudes differ. hemT is transcriptionally silent in 2.4.1 but actively transcribed in 2.4.9, and strongly upregulated under anaerobic-dark growth conditions when cells are respiring dimethyl sulfoxide, vs. aerobic-dark or phototrophic (anaerobic-light) conditions. Two extracytoplasmic function (ECF)-type sigma factors present in 2.4.9, but absent from 2.4.1 are directly involved in hemT transcription. Kinetic properties of the ALA synthases of all three strains were similar, but HemT enzymes are far less sensitive to feedback inhibition by hemin than HemA enzymes, and HemT is less active under oxidizing conditions. A model is presented that compares and contrast events in strains 2.4.1 and 2.4.9.
Assuntos
5-Aminolevulinato Sintetase/fisiologia , Ácido Aminolevulínico/metabolismo , Rhodobacter sphaeroides/enzimologia , 5-Aminolevulinato Sintetase/química , 5-Aminolevulinato Sintetase/genética , Proteínas de Bactérias/genética , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Genes BacterianosRESUMO
Biosynthesis of heme represents a complex process that involves multiple stages controlled by different enzymes. The first of these proteins is a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme, 5-aminolevulinate synthase (ALAS), that catalyzes the rate-limiting step in heme biosynthesis, the condensation of glycine with succinyl-CoA. Genetic mutations in human erythroid-specific ALAS (ALAS2) are associated with two inherited blood disorders, X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP). XLSA is caused by diminished ALAS2 activity leading to decreased ALA and heme syntheses and ultimately ineffective erythropoiesis, whereas XLPP results from "gain-of-function" ALAS2 mutations and consequent overproduction of protoporphyrin IX and increase in Zn2+-protoporphyrin levels. All XLPP-linked mutations affect the intrinsically disordered C-terminal tail of ALAS2. Our earlier molecular dynamics (MD) simulation-based analysis showed that the activity of ALAS2 could be regulated by the conformational flexibility of the active site loop whose structural features and dynamics could be changed due to mutations. We also revealed that the dynamic behavior of the two protomers of the ALAS2 dimer differed. However, how the structural dynamics of ALAS2 active site loop and C-terminal tail dynamics are related to each other and contribute to the homodimer asymmetry remained unanswered questions. In this study, we used bioinformatics and computational biology tools to evaluate the role(s) of the C-terminal tail dynamics in the structure and conformational dynamics of the murine ALAS2 homodimer active site loop. To assess the structural correlation between these two regions, we analyzed their structural displacements and determined their degree of correlation. Here, we report that the dynamics of ALAS2 active site loop is anti-correlated with the dynamics of the C-terminal tail and that this anti-correlation can represent a molecular basis for the functional and dynamic asymmetry of the ALAS2 homodimer.
Assuntos
5-Aminolevulinato Sintetase/química , Anemia Sideroblástica/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Heme/química , 5-Aminolevulinato Sintetase/genética , Anemia Sideroblástica/patologia , Animais , Domínio Catalítico , Biologia Computacional , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Heme/biossíntese , Heme/genética , Humanos , Camundongos , Simulação de Dinâmica Molecular , Mutação/genética , Multimerização Proteica/genéticaAssuntos
Anemia Sideroblástica/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , 5-Aminolevulinato Sintetase/química , 5-Aminolevulinato Sintetase/genética , Anemia Sideroblástica/tratamento farmacológico , Anemia Sideroblástica/genética , Sequência de Bases , Medula Óssea/metabolismo , Medula Óssea/patologia , Análise Mutacional de DNA , Transfusão de Eritrócitos , Doenças Genéticas Ligadas ao Cromossomo X/tratamento farmacológico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Hemizigoto , Hemoglobinas/análise , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Polimorfismo de Nucleotídeo Único , Piridoxina/uso terapêutico , Complexo Vitamínico B/uso terapêuticoRESUMO
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.
Assuntos
5-Aminolevulinato Sintetase/química , Ácido Aminolevulínico/química , Heme/química , Mitocôndrias/enzimologia , Subunidades Proteicas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Ácido Aminolevulínico/metabolismo , Domínio Catalítico , Clonagem Molecular , Coenzimas/química , Coenzimas/metabolismo , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Heme/biossíntese , Cinética , Mitocôndrias/química , Mitocôndrias/genética , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
The precursor to all tetrapyrroles is 5-aminolevulinic acid, which is made either via the condensation of glycine and succinyl-CoA catalyzed by an ALA synthase (the C4 or Shemin pathway) or by a pathway that uses glutamyl-tRNA as a precursor and involves other enzymes (the C5 pathway). Certain ALA synthases also catalyze the cyclization of ALA-CoA to form 2-amino-3-hydroxycyclopent-2-en-1-one. Organisms with synthases that possess this second activity nevertheless rely upon the C5 pathway to supply ALA for tetrapyrrole biosynthesis. The C5 N units are components of a variety of secondary metabolites. Here, we show that an ALA synthase used exclusively for tetrapyrrole biosynthesis is also capable of catalyzing the cyclization reaction, albeit at much lower efficiency than the dedicated cyclases. Two absolutely conserved serines present in all known ALA-CoA cyclases are threonines in all known ALA synthases, suggesting they could be important in distinguishing the functions of these enzymes. We found that purified mutant proteins having single and double substitutions of the conserved residues are not improved in their respective alternate activities; rather, they are worse. Protein structural modeling and amino acid sequence alignments were explored within the context of what is known about the reaction mechanisms of these two different types of enzymes to consider what other features are important for the two activities.
Assuntos
5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Substituição de Aminoácidos , Rhodobacter sphaeroides/enzimologia , 5-Aminolevulinato Sintetase/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Ciclização , Modelos Moleculares , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína , Tetrapirróis/biossíntese , Treonina/genéticaRESUMO
Heme biosynthesis, a complex, multistage, and tightly controlled process, starts with 5-aminolevulinate (ALA) production, which, in metazoa and certain bacteria, is a reaction catalyzed by 5-aminolevulinate synthase (ALAS), a pyridoxal 5'-phosphate (PLP)-dependent enzyme. Functional aberrations in ALAS are associated with several human diseases. ALAS can adopt open and closed conformations, with segmental rearrangements of a C-terminal, 16-amino acid loop and an α-helix regulating accessibility to the ALAS active site. Of the murine erythroid ALAS (mALAS2) forms previously engineered to assess the role of the flexible C-terminal loop versus mALAS2 function one stood out due to its impressive gain in catalytic power. To elucidate how the simultaneously introduced seven mutations of this activity-enhanced variant affected structural and dynamic properties of mALAS2, we conducted extensive molecular dynamics simulation analysis of the dimeric forms of wild-type mALAS2, hepta-variant and Rhodobacter capsulatus ALAS (aka R. capsulatus HemA). This analysis revealed that the seven simultaneous mutations in the C-terminal loop, which extends over the active site of the enzyme, caused the bacterial and murine proteins to adopt different conformations. Specifically, a new ß-strand in the mutated 'loop' led to interaction with two preexisting ß-strands and formation of an anti-parallel three-stranded ß-sheet, which likely endowed the murine hepta-variant a more 'stable' open conformation than that of wild-type mALAS2, consistent with a kinetic mechanism involving a faster closed-to-open conformation transition and product release for the mutated than wild-type enzyme. Further, the dynamic behavior of the mALAS2 protomers was strikingly different in the two dimeric forms.
Assuntos
5-Aminolevulinato Sintetase/química , Domínio Catalítico , Simulação de Dinâmica Molecular , Conformação Proteica , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Sequência de Aminoácidos , Animais , Biocatálise , Humanos , Cinética , Camundongos , Mutação , Rhodobacter capsulatus/enzimologia , Rhodobacter capsulatus/genética , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Mutations in the C-terminus of human erythroid 5-aminolevulinate synthase (hALAS2), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, are associated with two different blood disorders, X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP). XLSA-causing mutations yield hALAS2 variants with decreased activity, while XLPP-causing mutations result in a gain-of-function of hALAS2. There are no specific treatments for XLPP. Isonicotinic acid hydrazide (isoniazid, INH), an antituberculosis agent, can cause sideroblastic anemia as a side-effect, by limiting PLP availability to hALAS2, via inhibition of pyridoxal kinase or reaction with pyridoxal to form pyridoxal isonicotinoyl hydrazone. We hypothesized that INH also binds and directly inhibits hALAS2. Using fluorescence-activated cell sorting and confocal fluorescence microscopy, we demonstrate that INH reduces protoporphyrin IX levels in HeLa cells expressing either wild-type hALAS2 or XLPP variants. In addition, PLP and pyridoxamine 5'-phosphate (PMP) reversed the cellular inhibition of hALAS2 activity by INH. Steady-state kinetic analyses with purified hALAS2 indicated that INH directly inhibits the enzyme, noncompetitively or uncompetitively, with an apparent Ki of 1.2µM. Circular dichroism spectroscopy revealed that INH triggered tertiary structural changes in hALAS2 that altered the microenvironment of the PLP cofactor and hampered the association of PLP with apo-hALAS2. Treatment of four XLPP patients with INH (5mg·kg-1·day-1) over a six-month period was well tolerated but without statistically significant modification of PPIX levels. These results, taken together, permit us to further an INH inhibition kinetic mechanism for ALAS, which suggests the possible use of INH-derived drugs in treating patients with XLPP and potentially other protoporphyrin-accumulating porphyrias.
Assuntos
5-Aminolevulinato Sintetase/deficiência , Inibidores Enzimáticos/farmacologia , Doenças Genéticas Ligadas ao Cromossomo X/tratamento farmacológico , Isoniazida/farmacologia , Protoporfiria Eritropoética/tratamento farmacológico , 5-Aminolevulinato Sintetase/antagonistas & inibidores , 5-Aminolevulinato Sintetase/sangue , 5-Aminolevulinato Sintetase/química , 5-Aminolevulinato Sintetase/metabolismo , Anemia Sideroblástica/enzimologia , Inibidores Enzimáticos/uso terapêutico , Doenças Genéticas Ligadas ao Cromossomo X/sangue , Doenças Genéticas Ligadas ao Cromossomo X/enzimologia , Células HeLa , Humanos , Isoniazida/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Protoporfiria Eritropoética/sangue , Protoporfiria Eritropoética/enzimologia , Protoporfirinas/sangue , Fosfato de Piridoxal/metabolismo , Piridoxina/farmacologia , Complexo Vitamínico B/farmacologiaAssuntos
5-Aminolevulinato Sintetase/genética , Anemia Sideroblástica/genética , Genes Letais , Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação , 5-Aminolevulinato Sintetase/química , Sequência de Aminoácidos , Anemia Sideroblástica/diagnóstico , Medula Óssea/patologia , Análise Mutacional de DNA , Índices de Eritrócitos , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Mutação em Linhagem Germinativa , Heterozigoto , Humanos , Masculino , Modelos Moleculares , Linhagem , Fenótipo , Conformação ProteicaRESUMO
In this communication, we report the equilibrium and kinetic properties of the unfolding pathways of the native (pH 7.5) and alkaline molten globule (pH 10.5) states of the pyridoxal 5'-phosphate (PLP)-dependent enzyme 5-aminolevulinate synthase (ALAS). The stability of the molten globule state is adversely affected by thermal- and guanidine hydrochloride (GuHCl)-induced denaturation, and the equilibrium unfolding pathways, irrespective of pH, cannot be described with simple two-state models. Rapid kinetic measurements, in the presence of denaturing GuHCl concentrations, reveal that at pH 10.5, the rate of ALAS denaturation is 3 times faster than at pH 7.5. From pH jump experiments, comparable rates for the denaturation of the tertiary structure and PLP-microenvironment were discerned, indicating that the catalytic active site geometry strongly depends on the stable tertiary structural organization. Lastly, we demonstrate that partially folded ALAS tends to self-associate into higher oligomeric species at moderate GuHCl concentrations.