Vitamin B6 is the collective term for a group of three related compounds, pyridoxine (PN), pyridoxal (PL) and pyridoxamine (PM), and their phosphorylated derivatives, pyridoxine 5'-phosphate (PNP), pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP). Many enzymes are involved in the metabolism of vitamin B6, one of which is pyridoxine (pyridoxamine) 5’-phosphate oxidase (PPOX). PPOX has been well studied from bacteria, fungi and animals, but little research about PPOX has been done in plants.
In this study, two genes in the genome of Arabidopsis thaliana were found to encode for proteins that demonstrated PPOX activity: one gene is At5g49970 coding for a protein now designated AtPPOX-1, and the other gene is At2g46580 coding for a protein now designated AtPPOX-2. The cDNAs of At5g49970 and At2g46580 were cloned using the RT-PCR method, and then subcloned into the E. coli expression vector and subsequentely into yeast shuttle vector. PPOX functionally was demonstrated in two ways, i.e. demonstration of PPOX enzyme assay directly in vitro and by the complementation of a PDX3 (coding for yeast PPOX) knockout of yeast for increased oxidative stress susceptibility. However, the enzyme activity of AtPPOX-1 is almost 300-fold higher than that of AtPPOX-2. The inferred amino acid sequence of AtPPOX-1 is 530 amino acids long and that of AtPPOX-2 is 198 amino acids.
Although AtPPOX-1 and AtPPOX-2 are putatively isoenzymes, very little homology was found in their amino acid sequences. In order to understand their evolutionary relationship, phylogenetic analysis of AtPPOX-1 and AtPPOX-2 homologs across the three domains of life suggests that AtPPOX-1 and AtPPOX-2 have independent origins. AtPPOX-1 phylogeny appears congruent with the underlying branching pattern of the overall tree of life, while AtPPOX-2 phylogeny suggests that plant PPOX-2 may have originated from cyanobacteria. Presence of Yjef_N domain in land plants AtPPOX-1 homologs suggests that acquisition of this domain and its fusion with pyridox_oxidase domain began with the endosymbiotic acquisition of the chloroplast.
In this study, regulation of the AtPPOX-1 gene and its in vivo physiological functions were investigated. Variable levels of expression of AtPPOX-1 were seen in all tissues of A. thaliana examined. This gene is up-regulated by light, heat shock, ABA, JA and ethylene treatment, and down-regulated by exposure to NaCl. AtPPOX-1 may have two alternative splicing variants. One is expressed in all tissues, but the other is not expressed in root. To determine the biological role of AtPPOX-1, T-DNA insertional mutants of A. thaliana in AtPPOX-1 were analyzed. The mutant lines showed sensitivity to NaCl and high light for growth, and sensitivity to high concentrations of sucrose in cotyledon development. These results suggest involvement of AtPPOX-1 in stress tolerance and cotyledon development in A. thalia.