RESUMEN
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.
Asunto(s)
Enfermedad de Gaucher , Enfermedad de Niemann-Pick Tipo A , Enfermedades de Niemann-Pick , Niño , Humanos , Recién Nacido , Enfermedad de Niemann-Pick Tipo A/diagnóstico , Enfermedad de Niemann-Pick Tipo A/genética , Enfermedad de Gaucher/diagnóstico , Enfermedad de Gaucher/genética , Estudios Prospectivos , Enfermedades de Niemann-Pick/diagnóstico , Enfermedades de Niemann-Pick/genética , Esfingomielina Fosfodiesterasa/genética , Espectrometría de Masas en Tándem/métodosRESUMEN
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.
Asunto(s)
Biomarcadores/sangre , Enfermedad de Fabry/sangre , Glucolípidos/aislamiento & purificación , Esfingolípidos/aislamiento & purificación , Pruebas con Sangre Seca , Enfermedad de Fabry/genética , Enfermedad de Fabry/patología , Femenino , Glucolípidos/sangre , Humanos , Masculino , Mutación/genética , Esfingolípidos/sangreRESUMEN
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).
Asunto(s)
Tamizaje Neonatal , Prioridad del Paciente , Recién Nacido , Humanos , Inteligencia Artificial , Enfermedades Raras/diagnóstico , Enfermedades Raras/genética , Grupos FocalesRESUMEN
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.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/química , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C8 , Citocromo P-450 CYP2C9 , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
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.
Asunto(s)
Tamizaje Neonatal , Enfermedades Raras , Recién Nacido , Humanos , Niño , Tamizaje Neonatal/métodos , Enfermedades Raras/diagnóstico , Enfermedades Raras/epidemiología , Enfermedades Raras/genética , Inteligencia Artificial , Tecnología Digital , Europa (Continente)RESUMEN
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.
Asunto(s)
Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/metabolismo , Ingeniería Genética/métodos , Triticum/enzimología , Triticum/genética , Secuencia de Aminoácidos , Bacillus subtilis/genética , Biotecnología , Endo-1,4-beta Xilanasas/química , Activación Enzimática , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/métodos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Endo-1,4-beta Xilanasas/antagonistas & inhibidores , Inhibidores Enzimáticos/metabolismo , Proteínas de Plantas/metabolismo , Triticum/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Endo-1,4-beta Xilanasas/genética , Inhibidores Enzimáticos/química , Estabilidad de Enzimas , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Proteínas de Plantas/química , Proteínas de Plantas/genética , Temperatura , Triticum/química , Triticum/genéticaRESUMEN
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.
Asunto(s)
Endo-1,4-beta Xilanasas/antagonistas & inhibidores , Proteínas de Plantas/química , Proteínas de Plantas/farmacología , Triticum/enzimología , Alanina/metabolismo , Sustitución de Aminoácidos , Aspergillus niger/enzimología , Bacillus subtilis/enzimología , Dicroismo Circular , Endo-1,4-beta Xilanasas/clasificación , Endo-1,4-beta Xilanasas/metabolismo , Glutamina/metabolismo , Histidina/química , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Cinética , Lisina/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Pichia/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de Superficie , Trichoderma/enzimologíaRESUMEN
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.
Asunto(s)
Arabidopsis/citología , Arabidopsis/enzimología , Pared Celular/enzimología , beta-Fructofuranosidasa/química , Cristalización , Difracción de Rayos XRESUMEN
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.
Asunto(s)
Bacillus subtilis/enzimología , Endo-1,4-beta Xilanasas/química , Inhibidores Enzimáticos/química , Proteínas de Plantas/química , Triticum/química , Dominio Catalítico , Cristalografía por Rayos X , Endo-1,4-beta Xilanasas/antagonistas & inhibidores , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Mutación , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacología , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacología , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Homología Estructural de Proteína , Especificidad por Sustrato , Triticum/metabolismoRESUMEN
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.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/química , Cromanos/farmacología , Felodipino/farmacología , Tiazolidinedionas/farmacología , Tretinoina/farmacología , Acetatos/farmacología , Alitretinoína , Sitios de Unión , Cristalografía por Rayos X/métodos , Ciclopropanos , Citocromo P-450 CYP2C8 , Hemo/química , Humanos , Hierro/química , Antagonistas de Leucotrieno/farmacología , Ligandos , Conformación Molecular , Unión Proteica , Quinolinas/farmacología , Especificidad por Sustrato , Sulfuros , TroglitazonaRESUMEN
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.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/química , Hidrocarburo de Aril Hidroxilasas/ultraestructura , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/ultraestructura , Modelos Químicos , Modelos Moleculares , Sustitución de Aminoácidos , Hidrocarburo de Aril Hidroxilasas/genética , Sitios de Unión , Simulación por Computador , Citocromo P-450 CYP2A6 , Activación Enzimática , Humanos , Oxigenasas de Función Mixta/genética , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Benzoflavonas/metabolismo , Citocromo P-450 CYP1A2/química , Inhibidores Enzimáticos/metabolismo , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Citocromo P-450 CYP1A2/metabolismo , Inhibidores del Citocromo P-450 CYP1A2 , Humanos , Hidroxilación , Modelos Moleculares , Oxidación-Reducción , Conformación ProteicaRESUMEN
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.
Asunto(s)
Endo-1,4-beta Xilanasas/antagonistas & inhibidores , Proteínas de Plantas/química , Proteínas de Plantas/farmacología , Triticum/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , ADN/química , Cartilla de ADN/química , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Glutamina/química , Glicósido Hidrolasas/química , Modelos Genéticos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Mutagénesis Sitio-Dirigida , Pichia/metabolismo , Plásmidos/metabolismo , Mutación Puntual , Reacción en Cadena de la Polimerasa , Prolina/química , Isoformas de Proteínas , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Xilano Endo-1,3-beta-Xilosidasa/químicaRESUMEN
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.
Asunto(s)
Aspergillus niger/enzimología , Proteínas de Plantas/química , Xilosidasas/química , Sitios de Unión , Activación Enzimática , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Estructura Molecular , Proteínas de Plantas/metabolismo , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Especificidad por Sustrato , Xilosidasas/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Aspergillus niger/enzimología , Bacillus subtilis/enzimología , Proteínas Bacterianas , Endo-1,4-beta Xilanasas , Proteínas Fúngicas , Proteínas de Plantas , Triticum/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cristalización , Cristalografía por Rayos X , Endo-1,4-beta Xilanasas/química , Endo-1,4-beta Xilanasas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Unión ProteicaRESUMEN
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.
Asunto(s)
Cichorium intybus/enzimología , Glicósido Hidrolasas/química , Proteínas de Plantas/química , Cristalización , Cristalografía por Rayos X , Fructanos/metabolismo , Glicósido Hidrolasas/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
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.