Characterization of phosphofructokinase B-type kinases from Arabidopsis thaliana leading to the identification of a plastid inosine kinase

Abstract

Nucleotide metabolism is vital for plant development and dependent on the support of various enzymes including the phosphofructokinase B-type (PfkB) sugar kinases. Although some PfkB kinases such as adenosine kinase (ADK), ribokinase (RBSK), and pseudouridine kinase (PUKI) have been successfully identified and characterized in recent years, much is still unknown about these kinases. In this study, PfkB kinases from Arabidopsis thaliana were functionally screened leading to the identification of a plastid inosine kinase (PINK) that is involved in the feedback regulation of de novo purine synthesis and of kinase 6-2 (K6-2), which may be involved in negative regulation of purine synthesis. According to bioinformatic analyses, PINK is highly conserved in the plant kingdom, Subcellular localization and in vitro kinase activity analyses revealed that PINK is a plastid kinase that phosphorylates inosine, uridine, and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr). Furthermore, metabolite analysis was performed of Arabidopsis seedlings in which PINK expression was varied, which showed that PINK is not only involved in the inosine salvage but also likely contributes to feedback inhibition of de novo purine biosynthesis by regulating the plastid IMP pool. Moreover, PINK is possibly involved in pyrimidine nucleotide synthesis, at least in the context of defective uridine degradation. Additionally, another member of the PfkB family, kinase 6-2 (K6-2) was partially characterized in this work. K6-2 is localized in the chloroplast and in vitro biochemical analysis showed that K6-2 can phosphorylate AICAr, inosine, and guanosine. Furthermore, an in vivo functional study suggested that K6-2 is a negative regulator of purine de novo biosynthesis, as the biosynthesis of purine nucleotides was enhanced in the knock-down mutant of K6-2. Moreover, the reduced expression of K6-2 in Arabidopsis led to a phenotype that involved yellowing and suppressed leaf growth