Cloning, biochemical characterization and expression of a sunflower (Helianthus annuus L.) hexokinase associated with seed storage compounds accumulation
| Autor: | Enrique Martínez-Force, Rafael Garcés, Sonia Dorion, M.-C. Moisan, Jean Rivoal, Manuel Adrián Troncoso-Ponce |
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| Rok vydání: | 2011 |
| Předmět: |
DNA
Complementary Physiology Molecular Sequence Data Mannose Plant Science Gene Expression Regulation Enzymologic chemistry.chemical_compound Affinity chromatography Gene Expression Regulation Plant Hexokinase Expression levels Helianthus annuus Escherichia coli Amino Acid Sequence Cloning Molecular chemistry.chemical_classification Base Sequence Sequence Homology Amino Acid biology Biochemical characterization Seed Storage Proteins Temperature Active site Fructose Sequence Analysis DNA Metabolism Hydrogen-Ion Concentration Recombinant Proteins Protein Structure Tertiary Sunflower Hexoses metabolism Enzyme chemistry Biochemistry Seeds biology.protein Helianthus Sequence Alignment Agronomy and Crop Science |
| Zdroj: | Digital.CSIC. Repositorio Institucional del CSIC instname |
| ISSN: | 0176-1617 |
| Popis: | A full-length hexokinase cDNA, HaHXK1, was cloned and characterized from Helianthus annuus L. developing seeds. Based on its sequence and phylogenetic relationships, HaHXK1 is a membrane-associated (type-B) hexokinase. The predicted structural model resembles known hexokinase structures, folding into two domains of unequal size: a large and a small one separated by a deep cleft containing the residues involved in the enzyme active site. A truncated version, without the 24 N-terminal residues, was heterologously expressed in Escherichia coli, purified to electrophoretic homogeneity using immobilized metal ion affinity chromatography and biochemically characterized. The purified enzyme behaved as a monomer on size exclusion chromatography and had a specific activity of 19.3 μmol/min/mg protein, the highest specific activity ever reported for a plant hexokinase. The enzyme had higher affinity for glucose and mannose relative to fructose, but the enzymatic efficiency was higher with glucose. Recombinant HaHXK1 was inhibited by ADP and was insensitive either to glucose-6-phosphate or to trehalose-6-phosphate. Its expression profile showed higher levels in heterotrophic tissues, developing seeds and roots, than in photosynthetic ones. A time course of HXK activity and expression in seeds showed that the highest HXK levels are found at the early stages of reserve compounds, lipids and proteins accumulation. © 2010 Elsevier GmbH. This work was supported by Spanish Ministerio de Ciencia e Innovación and FEDER, project AGL2005-00100. This work was supported in part by a discovery grant from the Natural Sciences and Engineering Research Council of Canada to J. Rivoal. |
| Databáze: | OpenAIRE |
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