Cloning and characterization of a cDNA encoding hexokinase from tomato

Two different partial sequences encoding putative hexokinase (HXK, ATP: hex ose-6-phosphotransferase: EC 2.7.1.1) were isolated from tomato (Lycopersic on esculentum) by RT-PCR using degenerate primers. Southern blot analysis s uggested the existence of two divergent HXK genes. A complete cDNA of one H XK was isolated by screening a cDNA library prepared from young cherry toma to fruit. The 1770 bp cDNA of LeHXK2 contained an open reading frame encodi ng a 496 amino acid protein that has 69% identity with the two Arabidopsis HXKs, 83 and 85% identity with potato StHXK1 and tobacco NtHXK, respectivel y. However, this clone had 97% amino acid identity with potato StHXK2 and, therefore, was named LeHXK2. LeHXK2 cDNA was expressed in a triple mutant y east (Saccharomyces cerevisiae) strain which lacked the ability to phosphor ylate glucose and fructose and, therefore, was unable to grow on these suga rs as carbon sources. Mutant cells expressing LeHXK2 grew on both glucose a nd fructose with shorter doubling time on glucose. The kinetic properties o f LeHXK2 expressed in yeast were determined after the purification of LeHXK 2 by HPLC-ion exchange chromatography, confirming the identity of LeHXK2 as hexokinase with higher affinity to glucose. LeHXK2 mRNA was detected by RT -PCR expression analysis in all organs and tissues and at all stages of fru it development. However, semi-quantitative RT-PCR analysis showed that LeHX K2 was most highly expressed in flowers. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.