The Arabidopsis thaliana phosphate starvation responsive gene AtPPsPase1 encodes a novel type of inorganic pyrophosphatase

• Published in: Biochimica et biophysica acta Vol. 1810; no. 2; pp. 178 - 185
• Main Authors: May, Anett, Berger, Sigrid, Hertel, Thomas, Köck, Margret
• Format: Journal Article
• Language: English
• Published: Netherlands 01.02.2011
• Subjects: active sites; Amino Acid Sequence; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis Proteins - chemistry; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Arabidopsis thaliana; Biocatalysis - drug effects; Catalytic Domain; cytosol; Diphosphates - chemistry; Diphosphates - metabolism; Escherichia coli; gel chromatography; gene expression; Gene Expression Regulation, Enzymologic - drug effects; Gene Expression Regulation, Plant - drug effects; genes; homeostasis; Hydrogen-Ion Concentration; Immunoblotting; inorganic pyrophosphatase; Inorganic Pyrophosphatase - chemistry; Inorganic Pyrophosphatase - genetics; Inorganic Pyrophosphatase - metabolism; Kinetics; Magnesium - chemistry; Magnesium - metabolism; Magnesium - pharmacology; metabolism; Models, Molecular; molecular cloning; Molecular Sequence Data; Mutation; phosphates; Phosphates - metabolism; Phosphates - pharmacology; Protein Binding; Protein Folding; Protein Structure, Tertiary; recombinant proteins; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; Seedlings - enzymology; Seedlings - genetics; Sequence Homology, Amino Acid; Substrate Specificity
• ISSN: 0304-4165; 0006-3002
• DOI: 10.1016/j.bbagen.2010.11.006

Low inorganic phosphate (Pi) availability triggers metabolic responses to maintain the intracellular phosphate homeostasis in plants. One crucial adaptive mechanism is the immediate cleavage of Pi from phosphorylated substrates; however, phosphohydrolases that function in the cytosol and putative substrates have not been characterized yet. One candidate gene is Arabidopsis thaliana At1g73010 encoding an uncharacterized enzyme with homology to the haloacid dehalogenase (HAD) superfamily. This work reports the molecular cloning of At1g73010, its expression in Escherichia coli, and the enzymatic characterisation of the recombinant protein (33.5 kD). The Mg²(+)-dependent enzyme named AtPPsPase1 catalyzes the specific cleavage of pyrophosphate (K(m) 38.8 μM) with an alkaline catalytic pH optimum. Gel filtration revealed a tetrameric structure of the soluble cytoplasmic protein. Modelling of the active site and assay of the recombinant protein variant D19A demonstrated that the enzyme shares the catalytic mechanism of the HAD superfamily including a phosphorylated enzyme intermediate. The tight control of AtPPsPase1 gene expression underlines its important role in the Pi starvation response and suggests that cleavage of pyrophosphate is an immediate metabolic adaptation reaction. The novel enzyme, the first pyrophosphatase in the HAD superfamily, differs from classical pyrophosphatases with respect to structure and catalytic mechanism. The enzyme function could be used to discover unknown aspects of pyrophosphate metabolism in general.