Cloning and characterization of a sialidase from the filamentous fungus, Aspergillus fumigatus
| Autor: | Juliana H. F. Yeung, Andrew J. Bennet, Mark L. Warwas, Margo M. Moore, Arne Ø. Mooers, Deepani Indurugalla |
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| Rok vydání: | 2010 |
| Předmět: |
Glycan
Protein Conformation Molecular Sequence Data Neuraminidase medicine.disease_cause Sialidase Biochemistry Microbiology Aspergillus fumigatus Fungal Proteins chemistry.chemical_compound medicine Escherichia coli Amino Acid Sequence Cloning Molecular Molecular Biology Phylogeny chemistry.chemical_classification biology Base Sequence Cell Biology biology.organism_classification Fetuin N-Acetylneuraminic Acid Sialic acid Enzyme chemistry biology.protein N-Acetylneuraminic acid Hymecromone |
| Zdroj: | Glycoconjugate journal. 27(5) |
| ISSN: | 1573-4986 |
| Popis: | A gene encoding a putative sialidase was identified in the genome of the opportunistic fungal pathogen, Aspergillus fumigatus. Computational analysis showed that this protein has Asp box and FRIP domains, it was predicted to have an extracellular localization, and a mass of 42 kDa, all of which are characteristics of sialidases. Structural modeling predicted a canonical 6-bladed beta-propeller structure with the model's highly conserved catalytic residues aligning well with those of an experimentally determined sialidase structure. The gene encoding the putative Af sialidase was cloned and expressed in Escherichia coli. Enzymatic characterization found that the enzyme was able to cleave the synthetic sialic acid substrate, 4-methylumbelliferyl alpha-D-N-acetylneuraminic acid (MUN), and had a pH optimum of 3.5. Further kinetic characterization using 4-methylumbelliferyl alpha-D-N-acetylneuraminylgalactopyranoside revealed that Af sialidase preferred alpha2-3-linked sialic acids over the alpha2-6 isomers. No trans-sialidase activity was detected. qPCR studies showed that exposure to MEM plus human serum induced expression. Purified Af sialidase released sialic acid from diverse substrates such as mucin, fetuin, epithelial cell glycans and colominic acid, though A. fumigatus was unable to use either sialic acid or colominic acid as a sole source of carbon. Phylogenetic analysis revealed that the fungal sialidases were more closely related to those of bacteria than to sialidases from other eukaryotes. |
| Databáze: | OpenAIRE |
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