Cloning, biochemical characterization and expression of a sunflower (Helianthus annuus L.) hexokinase associated with seed storage compounds accumulation |
| |
Authors: | Troncoso-Ponce M A Rivoal J Dorion S Moisan M-C Garcés R Martínez-Force E |
| |
Affiliation: | a Instituto de la Grasa, CSIC, Avenida Padre Garcia Tejero 4, E-41012 Seville, Spain b Institut de Recherche en Biologie Végétale, Université de Montréal, 4101 Rue Sherbrooke est, Montréal, Que., Canada H1X 2B2 c Department of Plant Biology, Michigan State University, East Lansing, MI 48824-1312, USA |
| |
Abstract: | 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. |
| |
Keywords: | TAG, triacylglicerides HXK, hexokinase G6P, glucose-6-phosphate Ni-NTA, nickel-nitrilotriacetic acid LB, Luria Bertani IPTG, Isopropyl β- smallcaps" >d-1-thiogalactopyranoside T6P, trehalose-6-phosphate SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis ADP, adenosine diphosphate ATP, adenosine triphosphate UTP, uridine triphosphate CTP, cytidine triphosphate GTP, guanosine triphosphate |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|