Identification of a pyridoxine (pyridoxamine) 5'-phosphate oxidase from Arabidopsis thaliana.
Pyridoxine (pyridoxamine) 5'-phosphate oxidase (PPOX) catalyzes the oxidative conversion of pyridoxamine 5'-phosphate (PMP) or pyridoxine 5'-phosphate (PNP) to pyridoxal 5'-phosphate (PLP). The At5g49970 gene of Arabidopsis thaliana shows homology to PPOX's from a number of organisms including the Saccharomyces cerevisiae PDX3 gene. A cDNA corresponding to putative A. thaliana PPOX (AtPPOX) was obtained using reverse transcriptase-polymerase chain reaction and primers landing at the start and stop codons of At5g49970. The putative AtPPOX is 530 amino acid long and predicted to contain three distinct parts: a 64 amino acid long N-terminal putative chloroplast transit peptide, followed by a long Yjef_N domain of unknown function and a C-terminal Pyridox_oxidase domain. Recombinant proteins representing the C-terminal domain of AtPPOX and AtPPOX without transit peptide were expressed in E. coli and showed PPOX enzyme activity. The PDX3 knockout yeast deficient in PPOX activity exhibited sensitivity to oxidative stress. Constructs of AtPPOX cDNA of different lengths complemented the PDX3 knockout yeast for oxidative stress. The role of the Yjef_N domain of AtPPOX was not determined, but it shows homology with a number of conserved hypothetical proteins of unknown function.
Sang
Yuying
Y
Department of Biological Sciences, Auburn University, 101 Rouse Life Science Building, Auburn, AL 36849, USA.
Barbosa
Jose M
JM
Wu
Hongzhuan
H
Locy
Robert D
RD
Singh
Narendra K
NK
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D017343:Genes, Plant; D005816:Genetic Complementation Test; D018384:Oxidative Stress; D011734:Pyridoxaminephosphate Oxidase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D029681:Arabidopsis Proteins; D018744:DNA, Plant; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D011734:Pyridoxaminephosphate Oxidase