RESUMO
The clinical manifestation of sphingolipidosis leads often to misclassification between acid sphingomyelinase deficiency (ASMD) and Gaucher disease. In this multicenter, prospective study, we investigated a cohort of 31,838 individuals suspected to have Gaucher disease, due to clinical presentation, from 61 countries between 2017 and 2022. For all samples, both Acid-ß-glucocerebrosidase and acid sphingomyelinase enzyme activities were measured in dried blood spot specimens by tandem mass spectrometry followed by genetic confirmatory testing in potential positive cases. In total, 5933 symptomatic cases showed decreased enzyme activities and were submitted for genetic confirmatory testing. 1411/5933 (24%) cases were finally identified with Gaucher disease and 550/5933 (9%) with ASMD. Most of the confirmed ASMD cases were newborns and children below 2 years of age (63%). This study reveals that one in four cases suspected for Gaucher disease is diagnosed with ASMD. An early appropriate diagnostic work-up is essential because of the availability of a recently approved enzyme replacement therapy for ASMD. In conclusion, a diagnostic strategy using differential biochemical testing including genetic confirmatory testing in clinically suspected cases for sphingolipidosis is highly recommended.
Assuntos
Doença de Gaucher , Doença de Niemann-Pick Tipo A , Doenças de Niemann-Pick , Criança , Humanos , Recém-Nascido , Doença de Niemann-Pick Tipo A/diagnóstico , Doença de Niemann-Pick Tipo A/genética , Doença de Gaucher/diagnóstico , Doença de Gaucher/genética , Estudos Prospectivos , Doenças de Niemann-Pick/diagnóstico , Doenças de Niemann-Pick/genética , Esfingomielina Fosfodiesterase/genética , Espectrometria de Massas em Tandem/métodosRESUMO
A total of 11 948 females suspicious of Fabry disease were tested by a combined biochemical and genetic approach. The enzyme activity, together with the concentration of lyso-GL-3 (lyso-Gb3) biomarker in dried blood spots (DBS), substantially improved the diagnostic detection of Fabry disease in females compared to the enzyme activity alone. Abnormal values for both were highly suspicious of Fabry disease (97% positive predictive value [PPV], similar to PPV in males). In cases with one abnormal biochemical value, elevated lyso-GL-3 is a far more important indicator than low enzyme activity (39% PPV vs 6% PPV). Cases with clearly negative results for both biochemical parameters are unlikely to have Fabry disease, even in clinically highly suspicious cases.
Assuntos
Biomarcadores/sangue , Doença de Fabry/sangue , Glicolipídeos/isolamento & purificação , Esfingolipídeos/isolamento & purificação , Teste em Amostras de Sangue Seco , Doença de Fabry/genética , Doença de Fabry/patologia , Feminino , Glicolipídeos/sangue , Humanos , Masculino , Mutação/genética , Esfingolipídeos/sangueRESUMO
INTRODUCTION: Rare diseases (RDs) collectively impact over 30 million people in Europe. Most individual conditions have a low prevalence which has resulted in a lack of research and expertise in this field, especially regarding genetic newborn screening (gNBS). There is increasing recognition of the importance of incorporating patients' needs and general public perspectives into the shared decision-making process regarding gNBS. This study is part of the Innovative Medicine Initiative project Screen4Care which aims at shortening the diagnostic journey for RDs by accelerating diagnosis for patients living with RDs through gNBS and the use of digital technologies, such as artificial intelligence and machine learning. Our objective will be to assess expecting parent's perspectives, attitudes and preferences regarding gNBS for RDs in Italy and Germany. METHODS AND ANALYSIS: A mixed method approach will assess perspectives, attitudes and preferences of (1) expecting parents seeking genetic consultation and (2) 'healthy' expecting parents from the general population in two countries (Germany and Italy). Focus groups and interviews using the nominal group technique and ranking exercises will be performed (qualitative phase). The results will inform the treatment of attributes to be assessed via a survey and a discrete choice experiment (DCE). The total recruitment sample will be 2084 participants (approximatively 1000 participants in each country for the online survey). A combination of thematic qualitative and logit-based quantitative approaches will be used to analyse the results of the study. ETHICS AND DISSEMINATION: This study has been approved by the Erlangen University Ethics Committee (22-246_1-B), the Freiburg University Ethics Committee (23-1005 S1-AV) and clinical centres in Italy (University of FerraraCE: 357/2023/Oss/AOUFe and Hospedale Bambino Gesu: No.2997 of 2 November 2023, Prot. No. _902) and approved for data storage and handling at the Uppsala University (2022-05806-01). The dissemination of the results will be ensured via scientific journal publication (open access).
Assuntos
Triagem Neonatal , Preferência do Paciente , Recém-Nascido , Humanos , Inteligência Artificial , Doenças Raras/diagnóstico , Doenças Raras/genética , Grupos FocaisRESUMO
To identify the structural features underlying the distinct substrate and inhibitor profiles of P450 2C19 relative to the closely related human enzymes, P450s 2C8 and 2C9, the atomic structure (Protein Data Bank code 4GQS) of cytochrome P450 2C19 complexed with the inhibitor (2-methyl-1-benzofuran-3-yl)-(4-hydroxy-3,5-dimethylphenyl)methanone (Protein Data Bank chemical component 0XV) was determined to 2.87 Å resolution by x-ray crystallography. The conformation of the peptide backbone of P450 2C19 is most similar to that of P450 2C8, but the substrate-binding cavity of P450 2C8 is much larger than that of P450 2C19 due to differences in the amino acid residues that form the substrate-binding cavities of the two enzymes. In contrast, the substrate-binding cavity of P450 2C19 is much more similar in size to that of the structure of the P450 2C9 flurbiprofen complex than to that of a modified P450 2C9 or that of P450 2C8. The cavities of the P450 2C19 0XV complex and the P450 2C9 flurbiprofen complex differ, however, because the helix B-C loops of the two enzymes are dissimilar. These conformational differences reflect the effects of adjacent structural elements that interact with the B-C loops and that differ between the two enzymes. The availability of a structure for 2C19 will facilitate computational approaches for predictions of substrate and inhibitor binding to this enzyme.
Assuntos
Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C8 , Citocromo P-450 CYP2C9 , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
Since 72% of rare diseases are genetic in origin and mostly paediatrics, genetic newborn screening represents a diagnostic "window of opportunity". Therefore, many gNBS initiatives started in different European countries. Screen4Care is a research project, which resulted of a joint effort between the European Union Commission and the European Federation of Pharmaceutical Industries and Associations. It focuses on genetic newborn screening and artificial intelligence-based tools which will be applied to a large European population of about 25.000 infants. The neonatal screening strategy will be based on targeted sequencing, while whole genome sequencing will be offered to all enrolled infants who may show early symptoms but have resulted negative at the targeted sequencing-based newborn screening. We will leverage artificial intelligence-based algorithms to identify patients using Electronic Health Records (EHR) and to build a repository "symptom checkers" for patients and healthcare providers. S4C will design an equitable, ethical, and sustainable framework for genetic newborn screening and new digital tools, corroborated by a large workout where legal, ethical, and social complexities will be addressed with the intent of making the framework highly and flexibly translatable into the diverse European health systems.
Assuntos
Triagem Neonatal , Doenças Raras , Recém-Nascido , Humanos , Criança , Triagem Neonatal/métodos , Doenças Raras/diagnóstico , Doenças Raras/epidemiologia , Doenças Raras/genética , Inteligência Artificial , Tecnologia Digital , Europa (Continente)RESUMO
The Bacillus subtilis endoxylanase XynA (BSXY) is frequently used to improve the functionality of arabinoxylan-containing material in cereal based industries. The presence of endogenous Triticum aestivum xylanase inhibitors (TAXI-I and TAXI-II) in wheat is a real concern as they have a direct negative impact on the efficiency of this enzyme. Here, we used the recently determined structure of the complex between TAXI-I and an endoxylanase of Aspergillus niger to develop inhibitor-insensitive BSXY variants by site-directed mutagenesis of strategically chosen amino acids. We either induced steric hindrance to reject the inhibitors or interrupted key interactions with the inhibitors in the endoxylanase substrate-binding groove. The first strategy was successfully applied to position G12 where G12W combined inhibition insensitivity with unharmed catalytic performance. Variants from the second strategy showed altered inhibitor sensitivities concomitant with changes in enzyme activities and allowed to gain insight in the binding-mode of both TAXI-I and TAXI-II with BSXY.
Assuntos
Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Engenharia Genética/métodos , Triticum/enzimologia , Triticum/genética , Sequência de Aminoácidos , Bacillus subtilis/genética , Biotecnologia , Endo-1,4-beta-Xilanases/química , Ativação Enzimática , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/métodos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Two types of proteinaceous endoxylanase inhibitors occur in different cereals, i.e. the TAXI [Triticum aestivum endoxylanase inhibitor]-type and XIP [endoxylanase inhibiting protein]-type inhibitors. The present paper focuses on the TAXI-type proteins and deals with their structural characteristics and the identification, characterisation and heterologous expression of a TAXI gene from wheat. In addition, to shed light on the mechanism by which TAXI-type endoxylanase inhibitors work, the enzyme specificity, the optimal conditions for maximal inhibition activity, the molar complexation ratio and the inhibition kinetics of the inhibitors are explained and the effect of mutations of an endoxylanase on the inhibition by TAXIs is discussed.
Assuntos
Endo-1,4-beta-Xilanases/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Proteínas de Plantas/metabolismo , Triticum/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Endo-1,4-beta-Xilanases/genética , Inibidores Enzimáticos/química , Estabilidade Enzimática , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Temperatura , Triticum/química , Triticum/genéticaRESUMO
Wheat endoxylanase inhibitor TAXI-I inhibits microbial glycoside hydrolase family 11 endoxylanases. Crystallographic data of an Aspergillus niger endoxylanase-TAXI-I complex showed His374 of TAXI-I to be a key residue in endoxylanase inhibition. Its role in enzyme-inhibitor interaction was further investigated by site-directed mutagenesis of His374 into alanine, glutamine or lysine. Binding kinetics and affinities of the molecular interactions between A. niger, Bacillus subtilis, Trichoderma longibrachiatumendoxylanases and wild-type TAXI-I and TAXI-I His374 mutants were determined by surface plasmon resonance analysis. Enzyme-inhibitor binding was in accordance with a simple 1 : 1 binding model. Association and dissociation rate constants of wild-type TAXI-I towards the endoxylanases were in the range between 1.96 and 36.1 x 10(4)m(-1) x s(-1) and 0.72-3.60 x 10(-4) x s(-1), respectively, resulting in equilibrium dissociation constants in the low nanomolar range. Mutation of TAXI-I His374 to a variable degree reduced the inhibition capacity of the inhibitor mainly due to higher complex dissociation rate constants (three- to 80-fold increase). The association rate constants were affected to a smaller extent (up to eightfold decrease). Substitution of TAXI-I His374 therefore strongly affects the affinity of the inhibitor for the enzymes. In addition, the results show that His374 plays a critical role in the stabilization of the endoxylanase-TAXI-I complex rather than in the docking of inhibitor onto enzyme.
Assuntos
Endo-1,4-beta-Xilanases/antagonistas & inibidores , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Triticum/enzimologia , Alanina/metabolismo , Substituição de Aminoácidos , Aspergillus niger/enzimologia , Bacillus subtilis/enzimologia , Dicroísmo Circular , Endo-1,4-beta-Xilanases/classificação , Endo-1,4-beta-Xilanases/metabolismo , Glutamina/metabolismo , Histidina/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Cinética , Lisina/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Pichia/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Trichoderma/enzimologiaRESUMO
Cell-wall invertase 1 (AtcwINV1), a plant protein from Arabidopsis thaliana which is involved in the breakdown of sucrose, has been crystallized in two different crystal forms. Crystal form I grows in space group P3(1) or P3(2), whereas crystal form II grows in space group C222(1). Data sets were collected for crystal forms I and II to resolution limits of 2.40 and 2.15 A, respectively.
Assuntos
Arabidopsis/citologia , Arabidopsis/enzimologia , Parede Celular/enzimologia , beta-Frutofuranosidase/química , Cristalização , Difração de Raios XRESUMO
Triticum aestivum xylanase inhibitor (TAXI)-type inhibitors are active against microbial xylanases from glycoside hydrolase family 11, but the inhibition strength and the specificity towards different xylanases differ between TAXI isoforms. Mutational and biochemical analyses of TAXI-I, TAXI-IIA and Bacillus subtilis xylanase A showed that inhibition strength and specificity depend on the identity of only a few key residues of inhibitor and xylanase [Fierens K et al. (2005) FEBS J 272, 5872-5882; Raedschelders G et al. (2005) Biochem Biophys Res Commun335, 512-522; Sorensen JF & Sibbesen O (2006) Protein Eng Des Sel 19, 205-210; Bourgois TM et al. (2007) J Biotechnol 130, 95-105]. Crystallographic analysis of the structures of TAXI-IA and TAXI-IIA in complex with glycoside hydrolase family 11 B. subtilis xylanase A now provides a substantial explanation for these observations and a detailed insight into the structural determinants for inhibition strength and specificity. Structures of the xylanaseinhibitor complexes show that inhibition is established by loop interactions with active-site residues and substrate-mimicking contacts in the binding subsites. The interaction of residues Leu292 of TAXI-IA and Pro294 of TAXI-IIA with the -2 glycon subsite of the xylanase is shown to be critical for both inhibition strength and specificity. Also, detailed analysis of the interaction interfaces of the complexes illustrates that the inhibition strength of TAXI is related to the presence of an aspartate or asparagine residue adjacent to the acid/base catalyst of the xylanase, and therefore to the pH optimum of the xylanase. The lower the pH optimum of the xylanase, the stronger will be the interaction between enzyme and inhibitor, and the stronger the resulting inhibition.
Assuntos
Bacillus subtilis/enzimologia , Endo-1,4-beta-Xilanases/química , Inibidores Enzimáticos/química , Proteínas de Plantas/química , Triticum/química , Domínio Catalítico , Cristalografia por Raios X , Endo-1,4-beta-Xilanases/antagonistas & inibidores , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Mutação , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína , Especificidade por Substrato , Triticum/metabolismoRESUMO
Although a crystal structure and a pharmacophore model are available for cytochrome P450 2C8, the role of protein flexibility and specific ligand-protein interactions that govern substrate binding are poorly understood. X-ray crystal structures of P450 2C8 complexed with montelukast (2.8 A), troglitazone (2.7 A), felodipine (2.3 A), and 9-cis-retinoic acid (2.6 A) were determined to examine ligand-protein interactions for these chemically diverse compounds. Montelukast is a relatively large anionic inhibitor that exhibits a tripartite structure and complements the size and shape of the active-site cavity. The inhibitor troglitazone occupies the upper portion of the active-site cavity, leaving a substantial part of the cavity unoccupied. The smaller neutral felodipine molecule is sequestered with its dichlorophenyl group positioned close to the heme iron, and water molecules fill the distal portion of the cavity. The structure of the 9-cis-retinoic acid complex reveals that two substrate molecules bind simultaneously in the active site of P450 2C8. A second molecule of 9-cis-retinoic acid is located above the proximal molecule and can restrain the position of the latter for more efficient oxygenation. Solution binding studies do not discriminate between cooperative and noncooperative models for multiple substrate binding. The complexes with structurally distinct ligands further demonstrate the conformational adaptability of active site-constituting residues, especially Arg-241, that can reorient in the active-site cavity to stabilize a negatively charged functional group and define two spatially distinct binding sites for anionic moieties of substrates.
Assuntos
Hidrocarboneto de Aril Hidroxilases/química , Cromanos/farmacologia , Felodipino/farmacologia , Tiazolidinedionas/farmacologia , Tretinoína/farmacologia , Acetatos/farmacologia , Alitretinoína , Sítios de Ligação , Cristalografia por Raios X/métodos , Ciclopropanos , Citocromo P-450 CYP2C8 , Heme/química , Humanos , Ferro/química , Antagonistas de Leucotrienos/farmacologia , Ligantes , Conformação Molecular , Ligação Proteica , Quinolinas/farmacologia , Especificidade por Substrato , Sulfetos , TroglitazonaRESUMO
Human P450 2A6 displays a small active site that is well adapted for the oxidation of small planar substrates. Mutagenesis of CYP2A6 resulted in an increased catalytic efficiency for indole biotransformation to pigments and conferred a capacity to oxidize substituted indoles (Wu, Z.-L., Podust, L.M., Guengerich, F.P. J. Biol. Chem. 49 (2005) 41090-41100.). Here, we describe the structural basis that underlies the altered metabolic profile of three mutant enzymes, P450 2A6 N297Q, L240C/N297Q and N297Q/I300V. The Asn297 substitution abolishes a potential hydrogen bonding interaction with substrates in the active site, and replaces a structural water molecule between the helix B'-C region and helix I while maintaining structural hydrogen bonding interactions. The structures of the P450 2A6 N297Q/L240C and N297Q/I300V mutants provide clues as to how the protein can adapt to fit the larger substituted indoles in the active site, and enable a comparison with other P450 family 2 enzymes for which the residue at the equivalent position was seen to function in isozyme specificity, structural integrity and protein flexibility.
Assuntos
Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/ultraestrutura , Oxigenases de Função Mista/química , Oxigenases de Função Mista/ultraestrutura , Modelos Químicos , Modelos Moleculares , Substituição de Aminoácidos , Hidrocarboneto de Aril Hidroxilases/genética , Sítios de Ligação , Simulação por Computador , Citocromo P-450 CYP2A6 , Ativação Enzimática , Humanos , Oxigenases de Função Mista/genética , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Microsomal cytochrome P450 family 1 enzymes play prominent roles in xenobiotic detoxication and procarcinogen activation. P450 1A2 is the principal cytochrome P450 family 1 enzyme expressed in human liver and participates extensively in drug oxidations. This enzyme is also of great importance in the bioactivation of mutagens, including the N-hydroxylation of arylamines. P450-catalyzed reactions involve a wide range of substrates, and this versatility is reflected in a structural diversity evident in the active sites of available P450 structures. Here, we present the structure of human P450 1A2 in complex with the inhibitor alpha-naphthoflavone, determined to a resolution of 1.95 A. alpha-Naphthoflavone is bound in the active site above the distal surface of the heme prosthetic group. The structure reveals a compact, closed active site cavity that is highly adapted for the positioning and oxidation of relatively large, planar substrates. This unique topology is clearly distinct from known active site architectures of P450 family 2 and 3 enzymes and demonstrates how P450 family 1 enzymes have evolved to catalyze efficiently polycyclic aromatic hydrocarbon oxidation. This report provides the first structure of a microsomal P450 from family 1 and offers a template to study further structure-function relationships of alternative substrates and other cytochrome P450 family 1 members.
Assuntos
Benzoflavonas/metabolismo , Citocromo P-450 CYP1A2/química , Inibidores Enzimáticos/metabolismo , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Citocromo P-450 CYP1A2/metabolismo , Inibidores do Citocromo P-450 CYP1A2 , Humanos , Hidroxilação , Modelos Moleculares , Oxirredução , Conformação ProteicaRESUMO
Wheat grains contain Triticum aestivum xylanase inhibitor (TAXI) proteins which inhibit microbial xylanases, some of which are used in cereal based food industries. These inhibitors may play a role in plant defence. Among the TAXI isoforms described so far, TAXI-II displays a deviating inhibition specificity pattern. Here, we report on the molecular identity of TAXI-II and the basis of its inhibition specificity. Three candidate TAXI-II encoding sequences were isolated and recombinantly expressed in Pichia pastoris. To identify TAXI-II, the resulting proteins were tested against glycoside hydrolase family (GHF) 11 xylanases of Aspergillus niger (ANX) and Bacillus subtilis (BSX). One of these proteins (rTAXI-IB) inhibited both enzymes, like natural TAXI-I. The other candidates (rTAXI-IIA and rTAXI-IIB) showed an inhibition pattern typical for natural TAXI-II, only clearly inhibiting BSX. Comparative analysis of these highly similar sequences with distinct inhibition activity patterns, combined with information on the structural basis for ANX inhibition by TAXI-I [S. Sansen, C.J. De Ranter, K. Gebruers, K. Brijs, C.M. Courtin, J.A. Delcour, A. Rabijns, Structural basis for inhibition of Aspergillus niger xylanase by Triticum aestivum xylanase inhibitor-I, J. Biol. Chem. 279 (2004) 36022-36028], indicated a crucial role for Pro294 of TAXI-IIA and Gln376 of TAXI-IIB in determining the reduced inhibition activity towards ANX. Consequently, single point mutants rTAXI-IIA[P294L] and rTAXI-IIB[Q376H], both displaying the Leu/His combination corresponding to TAXI-I, were able to inhibit ANX. These results show that TAXI-II inhibition specificity bears on the identity of two key residues at positions 294 and 376, which are involved in the interaction at the -2 glycon subsite and the active site of GHF 11, respectively.
Assuntos
Endo-1,4-beta-Xilanases/antagonistas & inibidores , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Triticum/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , DNA/química , Primers do DNA/química , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Glutamina/química , Glicosídeo Hidrolases/química , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Mutagênese Sítio-Dirigida , Pichia/metabolismo , Plasmídeos/metabolismo , Mutação Puntual , Reação em Cadeia da Polimerase , Prolina/química , Isoformas de Proteínas , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Xilano Endo-1,3-beta-Xilosidase/químicaRESUMO
Plants developed a diverse battery of defense mechanisms in response to continual challenges by a broad spectrum of pathogenic microorganisms. Their defense arsenal includes inhibitors of cell wall-degrading enzymes, which hinder a possible invasion and colonization by antagonists. The structure of Triticum aestivum xylanase inhibitor-I (TAXI-I), a first member of potent TAXI-type inhibitors of fungal and bacterial family 11 xylanases, has been determined to 1.7-A resolution. Surprisingly, TAXI-I displays structural homology with the pepsin-like family of aspartic proteases but is proteolytically nonfunctional, because one or more residues of the essential catalytical triad are absent. The structure of the TAXI-I. Aspergillus niger xylanase I complex, at a resolution of 1.8 A, illustrates the ability of tight binding and inhibition with subnanomolar affinity and indicates the importance of the C-terminal end for the differences in xylanase specificity among different TAXI-type inhibitors.
Assuntos
Aspergillus niger/enzimologia , Proteínas de Plantas/química , Xilosidases/química , Sítios de Ligação , Ativação Enzimática , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Estrutura Molecular , Proteínas de Plantas/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Xilosidases/antagonistas & inibidoresRESUMO
Endo-beta-1,4-xylanases hydrolyze arabinoxylan, a major constituent of cereal cell walls, and are nowadays widely used in biotechnological processes. Purified complexes of family 11 xylanases from Aspergillus niger and Bacillus subtilis with TAXI I, a TAXI-type xylanase inhibitor from Triticum aestivum L., were prepared. In both cases the complex was crystallized using the hanging-drop vapour-diffusion method. The needle-like crystals of TAXI I in complex with A. niger xylanase belong to the trigonal space group P3(1) or P3(2), with unit-cell parameters a = b = 88.43, c = 128.99 A, and diffract to 1.8 A resolution. TAXI I in complex with B. subtilis xylanase crystallizes in the monoclinic space group C2, with a = 107.89, b = 95.33, c = 66.31 A, beta = 122.24 degrees. Complete data sets were collected for both crystal types using synchrotron radiation.
Assuntos
Aspergillus niger/enzimologia , Bacillus subtilis/enzimologia , Proteínas de Bactérias , Endo-1,4-beta-Xilanases , Proteínas Fúngicas , Proteínas de Plantas , Triticum/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalização , Cristalografia por Raios X , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ligação ProteicaRESUMO
Fructan 1-exohydrolase IIa (1-FEH IIa), a plant enzyme involved in fructan breakdown, has been crystallized using the hanging-drop vapour-diffusion method at 277 K. The crystals are tetragonal, belonging to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 139.83, b = 139.83, c = 181.94 A. Calculation of the Matthews coefficient indicates there to be two or three molecules in the asymmetric unit. Synchrotron radiation was used to collect a complete native data set to a resolution of 2.35 A.
Assuntos
Cichorium intybus/enzimologia , Glicosídeo Hidrolases/química , Proteínas de Plantas/química , Cristalização , Cristalografia por Raios X , Frutanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Proteínas de Plantas/metabolismoRESUMO
A TAXI-type endoxylanase inhibitor from T. aestivum L. wheat flour has been crystallized using the hanging-drop vapour-diffusion method. The needle-like crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 49.92, b = 66.45, c = 106.42 A. From these crystals, a native data set and a gold-derivative data set were collected to 2.25 and 1.75 A resolution, respectively. The heavy-atom derivative of this crystal form was obtained by the soaking method and allowed determination of the initial phases.