Identification of the Plant Ribokinase and Discovery of a Role for Arabidopsis Ribokinase in Nucleoside Metabolism

Abstract

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...

The ribokinases of Arabidopsis thaliana and Saccharomyces cerevisiae are required for ribose recycling from nucleotide catabolism, which in plants is not essential to survive prolonged dark stress

“…S5). Several proteomic studies (listed in the SUBA3 web resource; Hooper et al ., ) also found RBSK in the plastid stroma, and very recently At RBSK was shown by confocal microscopy to locate to nucleoids within the chloroplast (Riggs et al ., ), explaining our observation of plastid substructures labeled by RBSK‐YFP.…”

“…Every clade comprises sequences from all analyzed species. The RBSK candidates are present in clade 5 showing that these sequences indeed form a distinct group of orthologs, which confirms results very recently reported in another study (Riggs et al ., ). The clades are separated with high confidence, indicating that they represent distinct (enzymatic) functions of PfkB kinases, which are conserved in monocot and dicot species.…”

“…The apparent K M values for ribose of 85.4 and 55.3 μM for the Arabidopsis and yeast enzyme, respectively, were similar to the value reported for the enzyme from E. coli (110 μM; Maj & Gupta, ) and in the same order of magnitude as very recently reported for the enzyme from Arabidopsis (150 μM; Riggs et al ., ). Values for the apparent k cat for ribose were only reported for the enzyme of Leishmania major (10.8 s −1 , Ogbunude et al ., ) and very recently for RBSK from Arabidopsis (2 s −1 ; Riggs et al ., ), which, surprisingly, is c . 65 times lower than the value of 130.4 s −1 determined in this study.…”

“…Riggs et al . () used an At RBSK variant lacking the first 67 amino acids expressed in E. coli and noted that the correct N‐terminal processing of the enzyme is important for activity. We expressed the full‐length protein in a plant system and sequenced the purified protein processed in planta by mass spectrometry, demonstrating that the cleavage site for the transit peptide must lie upstream of amino acid 47 (Fig.…”

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

“…4g), suggesting that each of the seven enzymes possesses FRK activity and that their activity is specific for fructose. The two proteins that comprise the most closely related phylogenetic branch, FLN1 and FLN2, lack detectable fructokinase activity [23], and the only biochemically characterized enzyme in the unresolved polytomy of proteins related to the FRK clade, At1g17160, specifically phosphorylates ribose [28], suggesting that the seven FRKs identified herein likely comprise the Arabidopsis FRK family. As such, we propose the nomenclature in Table 1 based on previously defined nomenclature [23, 25] and degree of similarities among the Arabidopsis proteins, and we will use it in the remainder of this manuscript.

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