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Gloria C. Ferreira – One of the best experts on this subject based on the ideXlab platform.
Anti-Correlation between the Dynamics of the Active Site Loop and C-Terminal Tail in Relation to the Homodimer Asymmetry of the Mouse Erythroid 5-Aminolevulinate SynthaseMDPI AG, 2018Co-Authors: Dominique Catena, Gloria C. Ferreira, Min J. Kong, Vladimir N. UverskyAbstract:
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
Molecular dynamics analysis of the structural and dynamic properties of the functionally enhanced hepta-variant of mouse 5-Aminolevulinate Synthase.Journal of biomolecular structure & dynamics, 2017Co-Authors: Shelly Deforte, Gloria C. Ferreira, Bosko M. Stojanovski, Vladimir N. UverskyAbstract:
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 capsulatu…
The unfolding pathways of the native and molten globule states of 5-Aminolevulinate Synthase.Biochemical and biophysical research communications, 2016Co-Authors: Bosko M. Stojanovski, Vladimir N. Uversky, Leonid Breydo, Gloria C. FerreiraAbstract:
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.
Brian K. May – One of the best experts on this subject based on the ideXlab platform.
The major splice variant of human 5-Aminolevulinate Synthase-2 contributes significantly to erythroid heme biosynthesis.The international journal of biochemistry & cell biology, 2004Co-Authors: Timothy C. Cox, Sylvia S. Bottomley, Christopher S Matthews, Timothy J. Sadlon, Quenten Schwarz, Phillip D Wise, Liza L Cox, Brian K. MayAbstract:
Abstract The initial step of the heme biosynthetic pathway in erythroid cells is catalyzed by an erythroid-specific isoform of 5-Aminolevulinate Synthase-2 (ALAS2). Previously, an alternatively spliced mRNA isoform of ALAS2 was identified although the functional significance of the encoded protein was unknown. We sought to characterize the contribution of this ALAS2 isoform to overall erythroid heme biosynthesis. Here, we report the identification of three novel ALAS2 mRNA splice isoforms in addition to the previously described isoform lacking exon 4-derived sequence. Quantitation of these mRNAs using ribonuclease protection experiments revealed that the isoform without exon 4-derived sequence represents ∼35–45% of total ALAS2 mRNA while the newly identified transcripts together represent ∼15%. Despite the significant amounts of these three new transcripts, their features indicate that they are unlikely to substantially contribute to overall mitochondrial ALAS2 activity. In contrast, in vitro studies show that the major splice variant (lacking exon 4-encoded sequence) produces a functional enzyme, albeit with slightly reduced activity and with affinity for the ATP-specific, beta subunit of succinyl CoA Synthase, comparable to that of mature ALAS2. It was also established that the first 49 amino acids of the ALAS2 pre-protein are necessary and sufficient for translocation across the mitochondrial inner membrane and that this process is not affected by the absence of exon 4-encoded sequence. We conclude that the major splice isoform of ALAS2 is functional in vivo and could significantly contribute to erythroid heme biosynthesis and hemoglobin formation.
Regulation of erythroid 5-Aminolevulinate Synthase expression during erythropoiesis.The international journal of biochemistry & cell biology, 1999Co-Authors: Timothy J. Sadlon, Tania Dell’oso, Kathy H. Surinya, Brian K. MayAbstract:
Erythroid tissue is the major site of heme production in the body. The synthesis of heme and globin chains is coordinated at both the transcriptional and post-transcriptional levels to ensure that virtually no free heme or globin protein accumulates. The key rate-controlling enzyme of the heme biosynthetic pathway is 5-Aminolevulinate Synthase (ALAS) and an erythroid-specific isoform (ALAS2) is up-regulated during erythropoiesis. Differentiation of embryonic stem cells with a disrupted ALAS2 gene has established that expression of this gene is critical for erythropoiesis and cannot be compensated by expression of the ubiquitous isoform of the enzyme (ALAS1). Interestingly, heme appears to be important for expression of globin and other late erythroid genes and for erythroid cell differentiation although the mechanism of this effect is not clear. Transcriptional control elements that regulate the human gene for ALAS2 have been identified both in the promoter and in intronic enhancer regions. Subsequent translation of the ALAS2 mRNA is dependent on an adequate iron supply. The mechanism by which transcription of the gene for ALAS2 is increased by erythropoietin late in erythropoiesis remains an interesting issue. Erythropoietin action may result in altered levels of critical erythroid transcription factors or modulate the phosphorylation/acetylation status of these factors. Defects in the coding region of the gene for ALAS2 underlie the disease state X-linked sideroblastic anemia. In this review, we focus on the regulation and function of erythroid-specific 5-Aminolevulinate Synthase during erythropoiesis and its role in the X-linked sideroblastic anemia.
Phenobarbital-Induced Activation ofCYP2H1and 5-Aminolevulinate Synthase Genes in Chick Embryo Hepatocytes Is Blocked by an Inhibitor of Protein PhosphorylationArchives of biochemistry and biophysics, 1996Co-Authors: Satish C. Dogra, Brian K. MayAbstract:
Abstract The phenobarbital-induced activation of cytochrome P4502H1 ( CYP2H1 ) and 5-Aminolevulinate Synthase (ALAS-1) genes in chick embryo hepatocytes occurs at the level of gene transcription, but the molecular mechanism underlying this induction is not understood in detail. In the present study, we report that the protein kinase inhibitor 2-aminopurine markedly inhibits the phenobarbital-induced activation of CYP2H1 and ALAS-1 genes as measured by Northern blot analysis, but does not alter the basal expression of these genes in the absence of drug. Transient expression studies confirmed these findings. The construct pCATBg4.8 contains a 4.8-kb drug-responsive domain of the CYP2H1 gene fused to the enhancerless SV40 promoter and the drug-induced expression of this construct in chick embryo hepatocytes was inhibited by 2-aminopurine. Another construct pCAT, with the first 547 bp of 5′ flanking region of the CYP2H1 gene, is not responsive to drug and basal expression of this construct was not altered by the addition of 2-aminopurine. The evidence presented here demonstrates that the inhibitory action of 2-aminopurine on drug-induction is not due to a toxic effect on the cells. The induction of the CYP2H1 gene by phenobarbital was not altered by treating cells with the specific inhibitors for protein kinase C (GF 109203X and Ro 31-8220) or prolonged exposure to 12-0-tetradecanoyl- phorbol 13-acetate (TPA) or treatment with the specific inhibitors for tyrosine kinase (genistein and tyrphostin A25). Overall, the data indicate that a 2-aminopurine-sensitive protein kinase activity is required for the phenobarbital-induction mechanism but this is unlikely to be a protein kinase C or tyrosine kinase. It can be postulated that phosphorylation of a drug receptor protein may be an important step in the drug-induction process.
Gregory A. Hunter – One of the best experts on this subject based on the ideXlab platform.
Expression of Murine 5-Aminolevulinate Synthase Variants Causes Protoporphyrin IX Accumulation and Light-Induced Mammalian, 2016Co-Authors: Cell Death, Erica J. Fratz, Gregory A. Hunter, Gloria C. FerreiraAbstract:
5-Aminolevulinate Synthase (ALAS; EC 184.108.40.206) catalyzes the first committed step of heme biosynthesis in animals. The erythroid-specific ALAS isozyme (ALAS2) is negatively regulated by heme at the level of mitochondrial import and, in its mature form, certain mutations of the murine ALAS2 active site loop result in increased production of protoporphyrin IX (PPIX), the precursor for heme. Importantly, generation of PPIX is a crucial component in the widely used photodynamic therapies (PDT) of cancer and other dysplasias. ALAS2 variants that cause high levels of PPIX accumulation provide a new means of targeted, and potentially enhanced, photosensitization. In order to assess the prospective utility of ALAS2 variants in PPIX production for PDT, K562 human erythroleukemia cells and HeLa human cervical carcinoma cells were transfected with expression plasmids for ALAS2 variants with greater enzymatic activity than the wild-type enzyme. The levels of accumulated PPIX in ALAS2-expressing cells were analyzed using flow cytometry with fluorescence detection. Further, cells expressing ALAS2 variants were subjected to white light treatments (21–22 kLux) for 10 minutes after which cell viability was determined. Transfection of HeLa cells with expression plasmids for murine ALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of PPIX, particularly in the membrane. Light treatments revealed that ALAS2 expression results in an increase in cell death in comparison to aminolevulinic acid (ALA) treatment producing a similar amount of PPIX. The delivery of stable and highl
Human Erythroid 5-Aminolevulinate Synthase Mutations Associated with X-Linked Protoporphyria Disrupt the Conformational Equilibrium and Enhance Product Release.Biochemistry, 2015Co-Authors: Erica J. Fratz, Gregory A. Hunter, Vladimir N. Uversky, Leonid Breydo, Jerome Clayton, Sarah Ducamp, Jean-charles Deybach, Laurent Gouya, Hervé Puy, Gloria C. FerreiraAbstract:
Regulation of 5-Aminolevulinate Synthase (ALAS) is at the origin of balanced heme production in mammals. Mutations in the C-terminal region of human erythroid-specific ALAS (hALAS2) are associated with X-linked protoporphyria (XLPP), a disease characterized by extreme photosensitivity, with elevated blood concentrations of free protoporphyrin IX and zinc protoporphyrin. To investigate the molecular basis for this disease, recombinant hALAS2 and variants of the enzyme harboring the gain-of-function XLPP mutations were constructed, purified, and analyzed kinetically, spectroscopically, and thermodynamically. Enhanced activities of the XLPP variants resulted from increases in the rate at which the product 5-Aminolevulinate (ALA) was released from the enzyme. Circular dichroism spectroscopy revealed that the XLPP mutations altered the microenvironment of the pyridoxal 5′-phosphate cofactor, which underwent further and specific alterations upon succinyl-CoA binding. Transient kinetic analyses of the variant-ca…
Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-Aminolevulinate Synthase.Biochimica et biophysica acta, 2014Co-Authors: Bosko M. Stojanovski, Vladimir N. Uversky, Gregory A. Hunter, Leonid Breydo, Gloria C. FerreiraAbstract:
Abstract 5-Aminolevulinate Synthase (ALAS), a pyridoxal-5′phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in mammals. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0–3.0 and 7.5–10.5) and temperature (20 and 37 °C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH 2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH 10.5 and pH 9.5/37 °C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420 nm to 330 nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphthalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH 1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH 9.5/37 °C), although the kcat value is significantly decreased. Finally, the binding of 5-Aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme.