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1.
Protein J ; 42(5): 502-518, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37464145

RESUMO

The mechanism by which glycoside hydrolases control the reaction specificity through hydrolysis or transglycosylation is a key element embedded in their chemical structures. The determinants of reaction specificity seem to be complex. We looked for structural differences in domain B between the 4-α-glucanotransferase from Thermotoga maritima (TmGTase) and the α-amylase from Thermotoga petrophila (TpAmylase) and found a longer loop in the former that extends towards the active site carrying a W residue at its tip. Based on these differences we constructed the variants W131G and the partial deletion of the loop at residues 120-124/128-131, which showed a 11.6 and 11.4-fold increased hydrolysis/transglycosylation (H/T) ratio relative to WT protein, respectively. These variants had a reduction in the maximum velocity of the transglycosylation reaction, while their affinity for maltose as the acceptor was not substantially affected. Molecular dynamics simulations allow us to rationalize the increase in H/T ratio in terms of the flexibility near the active site and the conformations of the catalytic acid residues and their associated pKas.


Assuntos
Sistema da Enzima Desramificadora do Glicogênio , Thermotoga maritima , Hidrólise , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , alfa-Amilases , Especificidade por Substrato
2.
J Inherit Metab Dis ; 44(3): 534-543, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33141444

RESUMO

Adult polyglucosan body disease (APBD) represents a complex autosomal recessive inherited neurometabolic disorder due to homozygous or compound heterozygous pathogenic variants in GBE1 gene, resulting in deficiency of glycogen-branching enzyme and secondary storage of glycogen in the form of polyglucosan bodies, involving the skeletal muscle, diaphragm, peripheral nerve (including autonomic fibers), brain white matter, spinal cord, nerve roots, cerebellum, brainstem and to a lesser extent heart, lung, kidney, and liver cells. The diversity of new clinical presentations regarding neuromuscular involvement is astonishing and transformed APBD in a key differential diagnosis of completely different clinical conditions, including axonal and demyelinating sensorimotor polyneuropathy, progressive spastic paraparesis, motor neuronopathy presentations, autonomic disturbances, leukodystrophies or even pure myopathic involvement with limb-girdle pattern of weakness. This review article aims to summarize the main clinical, biochemical, genetic, and diagnostic aspects regarding APBD with special focus on neuromuscular presentations.


Assuntos
Sistema da Enzima Desramificadora do Glicogênio/genética , Doença de Depósito de Glicogênio/genética , Doença de Depósito de Glicogênio/fisiopatologia , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/fisiopatologia , Adulto , Encéfalo/patologia , Doença de Depósito de Glicogênio/patologia , Humanos , Músculo Esquelético/patologia , Doenças do Sistema Nervoso/patologia , Nervos Periféricos/patologia , Fenótipo , Medula Espinal/patologia
3.
Protein Pept Lett ; 27(2): 145-157, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31622193

RESUMO

BACKGROUND: Glycogen storage disease type III (GSDIII, Cori/Forbes disease) is a metabolic disorder due to the deficiency of the Glycogen Debranching Enzyme (GDE), a large monomeric protein (about 176 kDa) with two distinct enzymatic activities: 4-α-glucantransferase and amylo-α-1,6-glucosidase. Several mutations along the amylo-alpha-1,6-glucosidase,4-alphaglucanotransferase (Agl) gene are associated with loss of enzymatic activity. The unique treatment for GSDIII, at the moment, is based on diet. The potential of plants to manufacture exogenous engineered compounds for pharmaceutical purposes, from small to complex protein molecules such as vaccines, antibodies and other therapeutic/prophylactic entities, was shown by modern biotechnology through "Plant Molecular Farming". OBJECTIVE AND METHODS: In an attempt to develop novel protein-based therapeutics for GSDIII, the Agl gene, encoding for the human GDE (hGDE) was engineered for expression as a histidinetagged GDE protein both in Nicotiana benthamiana plants by a transient expression approach, and in axenic hairy root in vitro cultures (HR) from Lycopersicum esculentum and Beta vulgaris. RESULTS: In both plant-based expression formats, the hGDE protein accumulated in the soluble fraction of extracts. The plant-derived protein was purified by affinity chromatography in native conditions showing glycogen debranching activity. CONCLUSION: These investigations will be useful for the design of a new generation of biopharmaceuticals based on recombinant GDE protein that might represent, in the future, a possible therapeutic option for GSDIII.


Assuntos
Sistema da Enzima Desramificadora do Glicogênio/genética , Nicotiana/crescimento & desenvolvimento , Raízes de Plantas/citologia , Beta vulgaris/citologia , Beta vulgaris/genética , Beta vulgaris/metabolismo , Técnicas de Cultura de Células , Cromatografia de Afinidade , Regulação da Expressão Gênica de Plantas , Sistema da Enzima Desramificadora do Glicogênio/isolamento & purificação , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Humanos , Solanum lycopersicum/citologia , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/isolamento & purificação , Nicotiana/genética , Nicotiana/metabolismo
4.
Meat Sci ; 101: 83-9, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25462384

RESUMO

Muscle glycogen concentration (MGC) and lactate (LA), activity of glycogen debranching enzyme (GDE), glycogen phosphorylase (GP) and adenosine monophosphate kinase (AMPK) were determined at 0.5h (T0) and 24h (T24) post-mortem in Longissimus dorsi samples from 38 steers that produced high pH (>5.9) and normal pH (<5.8) carcasses at 24h postmortem. MGC, LA and glycolytic potential were higher (P<0.05) in normal pH carcasses. GDE activity was similar (P>0.05) in both pH categories. GP activity increased between T0 and T24 only in normal pH carcasses. AMPK activity was four times higher in normal pH v/s high pH carcasses, without changing its activity over time. Results reinforce the idea that differences in postmortem glycogenolytic/glycolytic flow in L. dorsi of steers showing normal v/s high muscle pH at 24h, could be explained not only by the higher initial MGC in normal pH carcasses, but also by a high and sustained activity of AMPK and an increased GP activity at 24h postmortem.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Glicogênio Fosforilase/metabolismo , Glicogênio/metabolismo , Glicólise/fisiologia , Carne/análise , Músculo Esquelético , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bovinos , Humanos , Concentração de Íons de Hidrogênio , Masculino , Músculo Esquelético/química , Músculo Esquelético/enzimologia , Fosforilação
5.
Semin Cell Dev Biol ; 21(5): 491-9, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20045480

RESUMO

The N-glycan-dependent quality control of glycoprotein folding prevents endoplasmic to Golgi exit of folding intermediates, irreparably misfolded glycoproteins and incompletely assembled multimeric complexes. It also enhances folding efficiency by preventing aggregation and facilitating formation of proper disulfide bonds. The control mechanism essentially involves four components, resident lectin-chaperones that recognize monoglucosylated polymannose glycans, a lectin-associated oxidoreductase acting on monoglucosylated glycoproteins, a glucosyltransferase that creates monoglucosytlated epitopes in protein-linked glycans and a glucosidase that removes the glucose units added by the glucosyltransferase. This last enzyme is the only mechanism component sensing glycoprotein conformations as it creates monoglucosylated glycans exclusively in not properly folded species or in not completely assembled complexes. The glucosidase is a dimeric heterodimer composed of a catalytic subunit and an additional one that is partially responsible for the ER localization of the enzyme and for the enhancement of the deglucosylation rate as its mannose 6-phosphate receptor homologous domain presents the substrate to the catalytic site. This review deals with our present knowledge on the glucosyltransferase and the glucosidase.


Assuntos
Glicoproteínas/química , Glicoproteínas/metabolismo , Animais , Cromatografia Gasosa , Glucose , Sistema da Enzima Desramificadora do Glicogênio , Complexo de Golgi/metabolismo , Humanos , Lectinas/metabolismo , Mananas , Chaperonas Moleculares , Polissacarídeos/metabolismo , Difosfato de Uridina/metabolismo , alfa-Glucosidases
6.
J Biomol Struct Dyn ; 22(1): 59-63, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15214806

RESUMO

Since the onset of the AIDS epidemic, some 20 million people have died and the estimate is that today close to 40 million are living with type 1 human immunodeficiency virus (HIV)/AIDS. About 14 thousands people are infected worldwide daily with this disease. Still, only a few pharmaceuticals are available for AIDS chemotheraphy. Some pharmaceuticals act against the virus before the entrance of the HIV into the host cells. One of these targets is the glucosidase protein. This class of enzymes has been recently explored because the synthesis of viral glycoproteins depends on the activity of enzymes, such as glucosidase and transferase, for the elaboration of the polysaccharides. In this work we study several glucosidase inhibitors. The DFT method is used to compute atomic charges and the ligand/receptor interaction was simulated with docking software. Analysis of the interactions of the proposed pharmaceutical, a pseudodisaccharide, with the Thermotoga maritima 4-alpha-glucanotransferase in complex with modified acarbose, the scores from docking as well as the graphical superposition of all the ligands, suggest that our molecular designed pseudo-disaccharide may be a potent glucosidase inhibitor.


Assuntos
Fármacos Anti-HIV/química , Desenho de Fármacos , Inibidores Enzimáticos/química , Glucosidases/antagonistas & inibidores , Acarbose/metabolismo , Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Fármacos Anti-HIV/uso terapêutico , Sítios de Ligação , Simulação por Computador , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Humanos , Modelos Moleculares
7.
Mol Plant Microbe Interact ; 15(4): 368-75, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12026175

RESUMO

The phosphoglucomutase (pgm) gene codes for a key enzyme required for the formation of UDP-glucose and ADP-glucose, the sugar donors for the biosynthesis of glucose containing polysaccharides. A Mesorhizobium loti pgm null mutant obtained in this study contains an altered form of lipopolysaccharide (LPS), lacks exopolysaccharide (EPS), beta cyclic glucan, and glycogen and is unable to nodulate Lotus tenuis. The nonnodulating phenotype of the pgm mutant was not due to the absence of glycogen, since a glycogen synthase (glgA) null mutant effectively nodulates this legume. In M. loti, pgm is part of the glycogen metabolism gene cluster formed by GlgP (glycogen phosphorylase), glgB (glycogen branching), glgC (ADP-glucose pyrophosphorylase), glgA, pgm, and glgX (glycogen debranching). The genes are transcribed as a single transcript from glgP to at least pgm under the control of a strong promoter (promoter I) upstream of glgP. An alternative promoter (promoter II), mapping in a 154-bp DNA fragment spanning 85 bp upstream of the glgA start codon and the first 69 bp of the glgA coding region, controls the expression of glgA and pgm, independently of the rest of the upstream genes. Primer extension experiments showed that transcription starts 19 bp upstream of the glgA start codon.


Assuntos
Glicogênio Sintase/genética , Glicogênio/metabolismo , Lotus/microbiologia , Fosfoglucomutase/genética , Rhizobiaceae/genética , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Agrobacterium tumefaciens/enzimologia , Agrobacterium tumefaciens/genética , Sequência de Bases , Genes Bacterianos/genética , Teste de Complementação Genética , Glucose-1-Fosfato Adenililtransferase , Sistema da Enzima Desramificadora do Glicogênio/genética , Glicogênio Fosforilase/genética , Óperon Lac/genética , Dados de Sequência Molecular , Mutação , Nucleotidiltransferases/genética , Óperon/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rhizobiaceae/enzimologia , Simbiose/genética , Transcrição Gênica
10.
Arch Invest Med (Mex) ; 22(3-4): 285-8, 1991.
Artigo em Inglês | MEDLINE | ID: mdl-1844114

RESUMO

An eleven year old boy was referred because of sudden loss of consciousness, muscular weakness, poor general health, severe hypoglycemia with seizures and hepatomegaly. Response to oral glucose and galactose increased blood lactic acid and glucose at different times. Fasting values of blood lactic was normal, but glucose was found at 33 mg/dl. Similar test made up two hours after feeding revealed hyperlactatemia (35-50 mg/dL) and hyperglycemia (129 mg/dL). Glucagon did not result in a rise of glucose at fasting or feeding. Hepatic glycogen content was found 15 gm/100 mg of tissue. The enzyme activities revealed a deficiency of the liver debranching enzyme while leukocytes had normal enzyme activity. Hepatic biopsy showed liver fibrosis. The present case had the clinical characteristics of severe form of glycogen storage disease. A low carbohydrate and high protein diet was indicated in order to increase the gluconeogenic precursors. Although debranching enzyme deficiency is almost always benign a high carbohydrate diet induced a more severe expression of the disease.


Assuntos
Carboidratos da Dieta/administração & dosagem , Proteínas Alimentares/administração & dosagem , Doença de Depósito de Glicogênio Tipo III/dietoterapia , Criança , Carboidratos da Dieta/efeitos adversos , Fibrose , Sistema da Enzima Desramificadora do Glicogênio/análise , Sistema da Enzima Desramificadora do Glicogênio/deficiência , Doença de Depósito de Glicogênio Tipo III/complicações , Doença de Depósito de Glicogênio Tipo III/enzimologia , Transtornos do Crescimento/etiologia , Humanos , Hipoglicemia/etiologia , Leucócitos/enzimologia , Fígado/enzimologia , Fígado/patologia , Glicogênio Hepático/metabolismo , Masculino , Músculos/patologia , Convulsões/etiologia
11.
J Pediatr ; 116(1): 95-100, 1990 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-2295969

RESUMO

To determine the tissue distribution of glycogen debranching enzyme, we used immunoblot analysis with a polyclonal antibody prepared against purified porcine muscle debranching enzyme. Debranching enzyme was identified in porcine brain, kidney, cardiac muscle, skeletal muscle, liver, and spleen; and in human liver, skeletal muscle, lymphocytes, lymphoblastoid cells, skin fibroblasts, cultured chorionic villi, and amniocytes. In each of these tissues the debranching enzyme band was 160 kd. To determine the molecular basis for glycogen storage disease type III at the protein level, tissues from 41 patients with glycogen storage disease type III were also subjected to immunoblot analysis. Three patients having isolated transferase deficiency with retention of glucosidase activity (type IIID disease) had nearly normal amounts of cross-reactive material. In the remaining patients (both transferase and glucosidase deficiency), debranching enzyme was either absent or greatly reduced. These latter patients included 31 with disease that appeared to involve both liver and muscle (type IIIA), four with disease that was present only in the liver (type IIIB), and three with unknown muscle status. In patients with both type IIIA and type IIIB disease, debranching enzyme protein was absent in skin fibroblasts, lymphoblastoid cells, and lymphocytes. The parents of two patients with type IIIA disease had an intermediate level of debranching enzyme protein, consistent with their presumed heterozygote state. An immunoblot analysis of cultured amniotic fluid cells from a woman whose fetus was at risk for type IIIA disease predicted an unaffected fetus; the prediction was confirmed postnatally. Thus Western blot analysis offers an alternate method of prenatal diagnosis for the most common form of glycogen storage disease type III.


Assuntos
Sistema da Enzima Desramificadora do Glicogênio/análise , Doença de Depósito de Glicogênio Tipo III/enzimologia , Doença de Depósito de Glicogênio/enzimologia , Western Blotting , Células Cultivadas , Feminino , Fibroblastos/enzimologia , Triagem de Portadores Genéticos , Doença de Depósito de Glicogênio Tipo III/classificação , Doença de Depósito de Glicogênio Tipo III/genética , Humanos , Fígado/enzimologia , Linfócitos/enzimologia , Músculos/enzimologia , Gravidez , Diagnóstico Pré-Natal
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