RESUMO
Vibrio vulnificus, a gram-negative bacterium, is the leading cause of seafood-borne illnesses and mortality in the United States. Previous studies have identified metabolites 2-C-methylerythritol 4-phosphate (MEP) as being essential for V. vulnificus growth and function. It was shown that 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr) is a critical enzyme in the viability of V. vulnificus, and many other bacteria, as it catalyzes the rearrangement of 1-deoxy-D-xylulose-5-phosphate (Dxp) to 2-C-methylerythritol 4-phosphate (MEP) within the MEP pathway, found in plants and bacteria. The MEP pathway produces the isoprenoids, isopentenyl diphosphate and dimethylallyl pyrophosphate. In this study, we produced and structurally characterized V. vulnificus Dxr. The enzyme forms a dimeric assembly and contains a metal ion in the active site. Protein produced in Escherichia coli co-purifies with Mg2+ ions, however the Mg2+ cations may be substituted with Mn2+, as both of these metals may be utilized by Dxrs. These findings will provide a basis for the design of Dxr inhibitors that may find application as antimicrobial compounds.
Assuntos
Aldose-Cetose Isomerases/química , Proteínas de Bactérias/química , Vibrio vulnificus/enzimologia , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Eritritol/análogos & derivados , Eritritol/metabolismo , Manganês/química , Manganês/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Fosfatos Açúcares/metabolismoRESUMO
Ragweed allergens affect several million people in the United States and Canada. To date, only two ragweed allergens, Amb t 5 and Amb a 11, have their structures determined and deposited to the Protein Data Bank. Here, we present structures of methylated ragweed allergen Amb a 8, Amb a 8 in the presence of poly(l-proline), and Art v 4 (mugwort allergen). Amb a 8 and Art v 4 are panallergens belonging to the profilin family of proteins. They share significant sequence and structural similarities, which results in cross-recognition by IgE antibodies. Molecular and immunological properties of Amb a 8 and Art v 4 are compared with those of Bet v 2 (birch pollen allergen) as well as with other allergenic profilins. We purified recombinant allergens that are recognized by patient IgE and are highly cross-reactive. It was determined that the analyzed allergens are relatively unstable. Structures of Amb a 8 in complex with poly(l-proline)10 or poly(l-proline)14 are the first structures of the plant profilin in complex with proline-rich peptides. Amb a 8 binds the poly(l-proline) in a mode similar to that observed in human, mouse, and P. falciparum profilin·peptide complexes. However, only some of the residues that form the peptide binding site are conserved.
Assuntos
Antígenos de Plantas/química , Imunoglobulina E/química , Animais , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Reações Cruzadas , Humanos , Imunoglobulina E/imunologia , Camundongos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Nonspecific lipid transfer proteins (nsLTPs) are basic proteins, stabilized by four disulfide bonds, and are expressed throughout the plant kingdom. These proteins are also known as important allergens in fruits and tree nuts. In this study, the nsLTP from hazelnuts, Cor a 8, was purified and its crystal structure determined. The protein is stable at low pH and refolds after thermal denaturation. Molecular dynamics simulations were used to provide an insight into conformational changes of Cor a 8 upon ligand binding. When known epitope areas from Pru p 3 were compared to those of Cor a 8, differences were obvious, which may contribute to limited cross-reactivity between peach and hazelnut allergens. Differences in epitope regions may contribute to limited cross-reactivity between Cor a 8 and nsLTPs from other plant sources. The structure of Cor a 8 represents the first resolved structure of a hazelnut allergen.
Assuntos
Alérgenos/química , Alérgenos/imunologia , Antígenos de Plantas/química , Antígenos de Plantas/imunologia , Corylus/química , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Reações Cruzadas , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologiaRESUMO
Kiwellin (Act d 5) is an allergenic protein contained in kiwifruit pulp in high amounts. The aim of this study was to investigate the three-dimensional structure of the natural molecule from green kiwifruit and its possible function. Kiwellin was crystallized, and its structure, including post-translational modifications, was elucidated. The molecular weight and structural features, in solution, were analyzed by gel filtration and circular dichroism, respectively. Although structurally similar to expansin, kiwellin lacks expansin activity and carbohydrate binding. A specific algorithm was applied to investigate any possible IgE reactivity correlation between kiwellin and a panel of 102 allergens, including expansins and other carbohydrate-binding allergens. The available data suggest a strong dependence of the kiwellin structure on the environmental/experimental conditions. This dependence therefore poses challenges in detecting the correlations between structural, functional, and immunological features of this protein.