Mutations in the FAD-binding fold of alcohol oxidase from Hansenula polymorpha
| Autor: | Geert Ab, Mieke Blaauw, M Dehoop, Marten Veenhuis, James M. Cregg, S Asgeirsdottir, M Gleeson |
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| Rok vydání: | 1991 |
| Předmět: |
Protein Conformation
Molecular Sequence Data Flavoprotein Bioengineering Biochemistry Pichia Structure-Activity Relationship Gene Expression Regulation Fungal Sequence Homology Nucleic Acid Enzyme Stability Histone octamer Amino Acid Sequence Site-directed mutagenesis Microscopy Immunoelectron Molecular Biology chemistry.chemical_classification Recombination Genetic Binding Sites biology Peroxisome Enzyme assay Alcohol oxidase Alcohol Oxidoreductases Enzyme chemistry FAD binding biology.protein Flavin-Adenine Dinucleotide Mutagenesis Site-Directed Biotechnology |
| Zdroj: | Protein engineering. 4(7) |
| ISSN: | 0269-2139 |
| Popis: | Alcohol oxidase of methylotrophic yeast is an FAD-containing enzyme. When in its active form, the enzyme is an octamer and located in the peroxisomes. To study the importance of FAD-binding on the activity, octamerization and intracellular localization of the enzyme, alcohol oxidase of Hansenula polymorpha was mutated in its presumed nucleotide-binding domain, which is formed by the N-terminal sequence. Whereas mutations of a glutamic acid residue (E42) reduced the stability of the octamer, it hardly affected enzyme activity and expression. However, replacements of three conserved glycines (G13, G15 and G18) and a conserved glutamic acid (E39) within the fold had severe effects. The mutations not only resulted in loss of enzyme activity but in reduced protein levels as well, probably due to decreased stability of the mutant alcohol oxidase. However, octamerization of the protein still occurred. The existence of inactive octameric proteins provides information about the formation pathway of this octameric flavoprotein. |
| Databáze: | OpenAIRE |
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