BackgroundFructose 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.ResultsProtein 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 μM. FRK7 was an outlier with a fructose Km of 12 μM. ATP binding was similar for all FRKs and ranged between 52 and 280 μM. 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.

Ribose can be used for energy or as a component of several important biomolecules, but for it to be used in either capacity it must first be phosphorylated by ribokinase (RBSK). RBSK proteins are part of the phosphofructokinase-B (pfkB) family of carbohydrate kinases. Ribose is a constituent of many critical biologically active molecules, including ATP and other energy information molecules, signaling molecules such as plant hormone cytokinins, metabolites such as NAD and S-AdoMet, and most notably nucleotides and their polymers RNA and DNA. Ribose can also be metabolized via the non-oxidative phase of the pentose phosphate pathway and glycolysis/TCA cycle to produce energy. In both prokaryotes and eukaryotes ribose is incorporated into these larger molecules or catabolized by initial conversion into ribose 5-phosphate via a phosphoryl transfer from ATP to the O5Ј position on ribose by ribokinase (RBSK) 2 (EC 2.7.1.15). No known major pathway utilizes unphosphorylated ribose. Indeed, the main source of ribose 5-phosphate is the isomerization of glucose 6-phosphate in the pentose phosphate pathway. As such, little is known about the significance of unphosphorylated ribose in cells or about the function of RBSK in plants.RBSK is a member of the ribokinase family of proteins, also known as the pfkB family (reviewed in Ref. 1), which includes several carbohydrate and nucleoside kinases. Although RBSK is likely conserved throughout evolution, as there are similar predicted proteins in genomes ranging from prokaryotes, to fungi, plants, and mammals, only bacterial and human RBSK proteins have been characterized biochemically (2-4). The structures of a handful of bacterial and human RBSKs have been solved, and the RBSK reaction mechanism is well understood (5, 6).The phosphorylation of ribose by RBSK requires the presence of both divalent and monovalent cations for catalysis and enzyme activation, respectively. Divalent cations, thought to be magnesium physiologically (7), aid in catalysis and form the metal-ATP chelate, which is the form bound by the enzyme. Monovalent cations, potassium physiologically, instead activate the Escherichia coli and human enzyme via an induced conformational change that forms an anion hole (4,7,8). Inorganic phosphate has been shown to activate both E. coli and human RBSK and other pfkB family proteins (4, 9, 10).The in vivo roles for RBSK activity vary dramatically by organism. Prokaryotes contain a ribose operon, encoding ribosebinding protein, PM-localized transporter, and RBSK (11), indicating a major function of this operon in utilization of extracellular ribose. Similarly for heterotrophic organisms, RSBK produces phosphorylated ribose for anabolic and catabolic reactions. In photosynthetic organisms, extracellular ribose might be a rare occurrence; hence the role of RSBK activity in these organisms is more obscure. In contrast to animals that primarily degrade nucleosides by phosphorolysis (12), plants begin nucleotide salvage and degradation by hydrolysis, releasing the nucleobase...