1,721,052 research outputs found
A molecular journey on the pathogenesis of primary hyperoxaluria
No full textPurpose of reviewPrimary hyperoxalurias (PHs) are rare disorders caused by the deficit of liver enzymes involved in glyoxylate metabolism. Their main hallmark is the increased excretion of oxalate leading to the deposition of calcium oxalate stones in the urinary tract. This review describes the molecular aspects of PHs and their relevance for the clinical management of patients.Recent findingsRecently, the study of PHs pathogenesis has received great attention. The development of novel in vitro and in vivo models has allowed to elucidate how inherited mutations lead to enzyme deficit, as well as to confirm the pathogenicity of newly-identified mutations. In addition, a better knowledge of the metabolic consequences in disorders of liver glyoxylate detoxification has been crucial to identify the key players in liver oxalate production, thus leading to the identification and validation of new drug targets.SummaryThe research on PHs at basic, translational and clinical level has improved our knowledge on the critical factors that modulate disease severity and the response to the available treatments, leading to the development of new drugs, either in preclinical stage or, very recently, approved for patient treatment
Cystalysin: an example of the catalytic versatility of pyridoxal 5'-phosphate dependent enzymes
No full textLiver peroxisomal alanine:glyoxylate aminotransferase and the effects of mutations associated with Primary Hyperoxaluria Type I: An overview.
No full textLiver peroxisomal alanine:glyoxylate aminotransferase (AGT) (EC 2.6.1.44) catalyses the conversion of l-alanine and glyoxylate to pyruvate and glycine, a reaction that allows glyoxylate detoxification. Inherited mutations on the AGXT gene encoding AGT lead to Primary Hyperoxaluria Type I (PH1), a rare disorder characterized by the deposition of calcium oxalate crystals primarily in the urinary tract. Here we describe the results obtained on the biochemical features of AGT as well as on the molecular and cellular effects of polymorphic and pathogenic mutations. A complex scenario on the molecular pathogenesis of PH1 emerges in which the co-inheritance of polymorphic changes and the condition of homozygosis or compound heterozygosis are two important factors that determine the enzymatic phenotype of PH1 patients. All the reported data represent relevant steps toward the understanding of genotype/phenotype correlations, the prediction of the response of the patients to the available therapies, and the development of new therapeutic approaches. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications
Folding pathway of the pyridoxal 5'-phosphate C-S lyase MalY from Escherichia coli
No full textMalY from Escherichia coli is a bifunctional dimeric PLP (pyridoxal
5-phosphate) enzyme acting as a β-cystathionase and as
a repressor of the maltose system. The spectroscopic and molecular
properties of the holoenzyme, in the untreated and NaBH4-
treated forms, and of the apoenzyme have been elucidated. A
systematic study of the urea-induced unfolding of MalY has
been monitored by gel filtration, cross-linking, ANS (8-anilino-
1-naphthalenesulphonic acid) binding and by visible, near- and
far-UV CD, fluorescence and NMR spectroscopies under equilibrium
conditions. Unfolding proceeds in at least three stages.
The first transition, occurring between 0 and 1 M urea, gives rise
to a partially active dimeric species that binds PLP. The second
equilibrium transition involving dimer dissociation, release of
PLP and loss of lyase activity leads to the formation of a monomeric
equilibrium intermediate. It is a partially unfolded molecule
that retains most of the native-state secondary structure,
binds significant amounts of ANS (a probe for exposed hydrophobic
surfaces) and tends to self-associate. The self-associated
aggregates predominate at urea concentrations of 2–4 M for
holoMalY. The third step represents the complete unfolding of
the enzyme. These results when compared with the urea-induced
unfolding profiles of apoMalY and NaBH4-reduced holoenzyme
suggest that the coenzyme group attached to the active-site lysine
residue increases the stability of the dimeric enzyme. Both holoand
apo-MalY could be successfully refolded into the active
enzyme with an 85% yield. Further refolding studies suggest
that large misfolded soluble aggregates that cannot be refolded
could be responsible for the incomplete re-activation
Natural and unnatural compounds rescue folding defects of human alanine:glyoxylate aminotransferase leading to Primary Hyperoxaluria Type I
No full textThe functional deficit of alanine:glyoxylate aminotransferase (AGT) in human hepatocytes leads to a rare recessive disorder named primary hyperoxaluria type I (PH1). PH1 is characterized by the progressive accumulation and deposition of calcium oxalate stones in the kidneys and urinary tract, leading to a life-threatening and potentially fatal condition. In the last decades, substantial progresses in the clarification of the molecular pathogenesis of the disease have been made. They resulted in the understanding that many mutations cause AGT deficiency by affecting the folding pathway of the protein leading to a reduced expression level, an increased aggregation propensity, and/or an aberrant mitochondrial localization. Thus, PH1 can be considered a misfolding disease and possibly treated by approaches aimed at counteracting the conformational defects of the variants. In this review, we summarize recent advances in the development of new strategies to identify molecules able to rescue AGT folding and trafficking either by acting as pharmacological chaperones or by preventing the mistargeting of the protein
Molecular insights into primary hyperoxaluria Type I pathogenesis.
No full textPrimary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disorder of glyoxylate metabolism caused by the deficiency of liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme. The PH1 pathogenesis is mostly due to single point mutations (more than 150 so far identified) on the AGXT gene, and is characterized by a marked heterogeneity in terms of genotype, enzymatic and clinical phenotypes. This article presents an up to date review of selected aspects of the biochemical properties of the two allelic forms of AGT and of some PH1-causing variants. These recent discoveries highlight the effects at the protein level of the pathogenic mutations, and, together with previous cell biology and clinical data, (i) improve the understanding of the molecular basis of PH1 pathogenesis, and (ii) help to delineate perspectives for predicting the response to pyridoxine treatment or for suggesting new strategies for PH1 patients bearing the analyzed mutations
Site-directed mutagenesis provides insight into racemization of alanine catlayzed by "Treponema denticola" cystalysin
No full textIn addition to alpha, beta-elimination of L-cysteine, Treponema denticola cystalysin catalyzes the racemization of both enantiomers of alanine accompanied by an overall transamination. Lys-238 and Tyr-123 or a water molecule located on the si and re face of the cofactor, respectively, have been proposed to act as the acid/base catalysts in the proton abstraction/donation at Calpha/C4' of the external aldimine. In this investigation, two site-directed mutants, K238A and Y123F, have been characterized. The Lys --> Ala mutation results in the complete loss of either lyase activity or racemase activity in both directions or transaminase activity toward L-alanine. However, the K238A mutant is able to catalyze the overall transamination of D-alanine, and only D-alanine is the product of the reverse transamination. For Y123F the k(cat)/K(m) is reduced 3.5-fold for alpha, beta-elimination, whereas it is reduced 300-400-fold for racemization. Y123F has approximately 18% of wild type transaminase activity with L-alanine and an extremely low transaminase activity with D-alanine. Moreover, the catalytic properties of the Y124F and Y123F/Y124F mutants rule out the possibility that the residual racemase and transaminase activities displayed by Y123F are due to Tyr-124. All these data, together with computational results, indicate a two-base racemization mechanism for cystalysin in which Lys-238 has been unequivocally identified as the catalyst acting on the si face of the cofactor. Moreover, this study highlights the importance of the interaction of Tyr-123 with water molecules for efficient proton abstraction/donation function on the re face
Characterization of C-S Lyase from C. diphtheriae: A Possible Target for New Antimicrobial Drugs
Full text linkThe emergence of antibiotic resistance in microbial pathogens requires the identification of new antibacterial drugs.The biosynthesis of methionine is an attractive target because of its central importance in cellular metabolism. Moreover, most of the steps in methionine biosynthesis pathway are absent in mammals, lowering the probability of unwanted side effects. Herein, detailed
biochemical characterization of one enzyme required formethionine biosynthesis, a pyridoxal-phosphate (PLP) dependent C-S lyase fromCorynebacterium diphtheriae, a pathogenic bacterium that causes diphtheria, has been performed.We overexpressed the
protein in E. coli and analyzed substrate specificity, pH dependence of steady state kinetic parameters, and ligand-induced spectral
transitions of the protein. We used site-directed mutagenesis to highlight the importance of active site residues Tyr55, Tyr114, and Arg351,analyzing the effects of amino acid replacement on catalytic properties of enzyme. Better understanding of the active site of C.
diphtheriae C-S lyase and the determinants of substrate and reaction specificity from this work will facilitate the design of novel
inhibitors as antibacterial therapeutics
The chaperone role of the pyridoxal 5′-phosphate and its implications for rare diseases involving B6-dependent enzymes
No full textThe biologically active form of the B6 vitamers is pyridoxal 5'-phosphate (PLP), which plays a coenzymatic role in several distinct enzymatic activities ranging from the synthesis, interconversion and degradation of amino acids to the replenishment of one-carbon units, synthesis and degradation of biogenic amines, synthesis of tetrapyrrolic compounds and metabolism of amino-sugars. In the catalytic process of PLP-dependent enzymes, the substrate amino acid forms a Schiff base with PLP and the electrophilicity of the PLP pyridine ring plays important roles in the subsequent catalytic steps. While the essential role of PLP in the acquisition of biological activity of many proteins is long recognized, the finding that some PLP-enzymes require the coenzyme for refolding in vitro points to an additional role of PLP as a chaperone in the folding process. Mutations in the genes encoding PLP-enzymes are causative of several rare inherited diseases. Patients affected by some of these diseases (AADC deficiency, cystathionuria, homocystinuria, gyrate atrophy, primary hyperoxaluria type 1, xanthurenic aciduria, X-linked sideroblastic anaemia) can benefit, although at different degrees, from the administration of pyridoxine, a PLP precursor. The effect of the coenzyme is not limited to mutations that affect the enzyme-coenzyme interaction, but also to those that cause folding defects, reinforcing the idea that PLP could play a chaperone role and improve the folding efficiency of misfolded variants. In this review, recent biochemical and cell biology studies highlighting the chaperoning activity of the coenzyme on folding-defective variants of PLP-enzymes associated with rare diseases are presented and discussed
Lysine 238 is an essential residue for alfa, beta- elimination catalyzed by "Treponema denticola" cystalysin
No full textTreponema denticola cystalysin is a pyridoxal 5′-phosphate (PLP) enzyme that catalyzes the α,β-elimination of L-cysteine to pyruvate, ammonia, and H2S. Similar to other PLP enzymes, an active site Lys residue (Lys-238) forms an internal Schiff base with PLP. The mechanistic role of this residue has been studied by an analysis of the mutant enzymes in which Lys-238 has been replaced by Ala (K238A) and Arg (K238R). Both apomutants reconstituted with PLP bind noncovalently ∼50% of the normal complement of the cofactor and have a lower affinity for the coenzyme than that of wild-type. Kinetic analyses of the reactions of K238A and K238R mutants with glycine compared with that of wild-type demonstrate the decrease of the rate of Schiff base formation by 103- and 7.5 × 104-fold, respectively, and, to a lesser extent, a decrease of the rate of Schiff base hydrolysis. Thus, a role of Lys-238 is to facilitate formation of external aldimine by transimination. Kinetic data reveal that the K238A mutant is inactive in the α,β-elimination of L-cysteine and β-chloro-L-alanine, whereas K238R retains 0.3% of the wild-type activity. These data, together with those derived from a spectral analysis of the reaction of Lys-238 mutants with unproductive substrate analogues, indicate that Lys-238 is an essential catalytic residue, possibly participating as a general base abstracting the Cα-proton from the substrate and possibly as a general acid protonating the β-leaving group
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