Identification and characterization of a pyridoxal 5′-phosphate phosphatase in tobacco plants

Abstract

Pyridoxal 5′-phosphate (PLP), the catalytically active form of vitamin B6, is an important cofactor for many biochemical transformations. PLP is also a very reactive molecule, and the most well-established mechanism for maintaining low levels of free PLP is its dephosphorylation by phosphatases. In our previous study, the crude enzyme extract from tobacco leaves rapidly hydrolyzed PLP at a pH optimum of 5.5. Using PLP as a substrate, a novel acid phosphatase was purified from tobacco leaves and characterized. Whether there is a PLP specific phosphatase in plants is still unknown. In this study, a cDNA clone sharing 34.72% homology with human PLP phosphatase sequences was identified from N. tabacum and characterized. The cDNA encodes a polypeptide of 319 amino acid residues, and the recombinant enzyme purified from E. coli exhibited maximum catalytic activity for PLP at pH 7.5. The properties of the purified enzyme, including pH optimum, metal requirement, optimum substrate and inhibitors were similar to those of human PLP phosphatase. Subcellular localization analysis showed that the PLP phosphatase is mainly located in chloroplast. We down-regulated the gene expression with plant RNA interference technology and found that the down-regulation has a greater impact on the transcription of genes encoding vitamin B6 metabolic enzymes. Our study further suggested that the PLP phosphatase plays an important role for maintaining PLP homeostasis within the chloroplast in plants.