Gene cloning and comparative analysis of pyridoxal 5′-phosphate salvage synthesis enzymes in tobacco plants

Abstract

Pyridoxal 5′-phosphate (PLP), the catalytically active form of vitamin B6, is an important cofactor for many biochemical transformations. Plants are able to synthesize PLP de novo, but they also have a salvage pathway that functions to convert different vitamer forms between each other. Although the salvage enzymes are identified successively from plants, many questions remain unanswered. In the salvage pathway, PLP is synthesized by an ATP-dependent pyridoxal kinase (PLK) and an FMN-dependent pyridoxine 5′-phosphate oxidase (PNPO). In this study, cDNAs encoding PLK and PNPO were cloned from tobacco plants, compared and analyzed, and then the gene expression was down-regulated by RNA interference. Our results show that the NtPLK and NtPNPO contains highly conserved motifs involved in substrate binding or catalysis. NtPLK and NtPNPO are essential enzymes for all tissues of tobacco plants, and NtPNPO is mainly located in chloroplast. The down-regulation of NtPLK and NtPNPO has a greater impact on the transcription level of other vitamin B6 metabolic enzymes. Based on the results, we speculate that, in plants, the de novo synthesized free PLP may be hydrolyzed to PL by phosphatase in cytosol, and then the PL enter into organelles. In organelles, salvage pathway plays its role to produce PLP for maintenance of vitamin B6-mediated processes.