Identification and biochemical characterization of the fructokinase gene family in Arabidopsis thaliana

Background: Fructose is an abundant sugar in plants as it is a breakdown product of both major sucrose-cleaving enzymes. To enter metabolism, fructose is phosphorylated by a fructokinase (FRK). Known FRKs are members of a diverse family of carbohydrate/purine kinases known as the phosphofructokinase B (pfkB) family. The complete complement of active fructokinases has not been reported for any plant species. Results: Protein sequence analysis of the 22 Arabidopsis thaliana pfkB members identified eight highly related predicted proteins, including one with previously demonstrated FRK activity. For one, At1g50390, the predicted open reading frame is half the size of active FRKs, and only incompletely spliced RNAs were identified, which led to a premature stop codon, both indicating that this gene does not produce active FRK. The remaining seven proteins were expressed in E. coli and phosphorylated fructose specifically in vitro leading us to propose a unifying nomenclature (FRK1-7). Substrate inhibition was observed for fructose in all FRKs except FRK1. Fructose binding was on the same order of magnitude for FRK1-6, between 260 and 480 mu M. FRK7 was an outlier with a fructose Km of 12 mu M. ATP binding was similar for all FRKs and ranged between 52 and 280 mu M. YFP-tagged AtFRKs were cytosolic, except plastidic FRK3. T-DNA alleles with non-detectable wild-type RNAs in five of the seven active FRK genes produced no overt phenotype. We extended our sequence comparisons to include putative FRKs encoded in other plant sequenced genomes. We observed that different subgroups expanded subsequent to speciation. Conclusions: Arabidopsis thaliana as well as all other plant species analyzed contain multiple copies of genes encoding FRK activity. Sequence comparisons among multiple species identified a minimal set of three distinct FRKs present on all species investigated including a plastid-localized form. The selective expansion of specific isozymes results in differences in FRK gene number among species. AtFRKs exhibit substrate inhibition, typical of their mammalian counterparts with the single AtFRK1 lacking this property, suggesting it may have a distinct in vivo role. Results presented here provide a starting point for the engineering of specific FRKs to affect biomass production.