Publications

• Active site structure and stereospecificity of Escherichia coli pyridoxine-5'-phosphate oxidase.

  di Salvo M.L., Ko T.-P., Musayev F.N., Raboni S., Schirch V., Safo M.K.

  Pyridoxine-5'-phosphate oxidase catalyzes the oxidation of either the C4' alcohol group or amino group of the two substrates pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate to an aldehyde, forming pyridoxal 5'-phosphate. A hydrogen atom is removed from C4' during the oxidation and a pair of ... >> More

  Pyridoxine-5'-phosphate oxidase catalyzes the oxidation of either the C4' alcohol group or amino group of the two substrates pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate to an aldehyde, forming pyridoxal 5'-phosphate. A hydrogen atom is removed from C4' during the oxidation and a pair of electrons is transferred to tightly bound FMN. A new crystal form of the enzyme in complex with pyridoxal 5'-phosphate shows that the N-terminal segment of the protein folds over the active site to sequester the ligand from solvent during the catalytic cycle. Using (4'R)-[(3)H]PMP as substrate, nearly 100 % of the radiolabel appears in water after oxidation to pyridoxal 5'-phosphate. Thus, the enzyme is specific for removal of the proR hydrogen atom from the prochiral C4' carbon atom of pyridoxamine 5'-phosphate. Site mutants were made of all residues at the active site that interact with the oxygen atom or amine group on C4' of the substrates. Other residues that make interactions with the phosphate moiety of the substrate were mutated. The mutants showed a decrease in affinity, but exhibited considerable catalytic activity, showing that these residues are important for binding, but play a lesser role in catalysis. The exception is Arg197, which is important for both binding and catalysis. The R197 M mutant enzyme catalyzed removal of the proS hydrogen atom from (4'R)-[(3)H]PMP, showing that the guanidinium side-chain plays an important role in determining stereospecificity. The crystal structure and the stereospecificity studies suggests that the pair of electrons on C4' of the substrate are transferred to FMN as a hydride ion. << Less

  J. Mol. Biol. 315:385-397(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Kinetic limitation and cellular amount of pyridoxine (pyridoxamine) 5'-phosphate oxidase of Escherichia coli K-12.

  Zhao G., Winkler M.E.

  We report the purification and enzymological characterization of Escherichia coli K-12 pyridoxine (pyridoxamine) 5'-phosphate (PNP/PMP) oxidase, which is a key committed enzyme in the biosynthesis of the essential coenzyme pyridoxal 5'-phosphate (PLP). The enzyme encoded by pdxH was overexpressed ... >> More

  We report the purification and enzymological characterization of Escherichia coli K-12 pyridoxine (pyridoxamine) 5'-phosphate (PNP/PMP) oxidase, which is a key committed enzyme in the biosynthesis of the essential coenzyme pyridoxal 5'-phosphate (PLP). The enzyme encoded by pdxH was overexpressed and purified to electrophoretic homogeneity by four steps of column chromatography. The purified PdxH enzyme is a thermally stable 51-kDa homodimer containing one molecule of flavin mononucleotide (FMN). In the presence of molecular oxygen, the PdxH enzyme uses PNP or PMP as a substrate (Km = 2 and 105 microM and kcat = 0.76 and 1.72 s-1 for PNP and PMP, respectively) and produces hydrogen peroxide. Thus, under aerobic conditions, the PdxH enzyme acts as a classical monofunctional flavoprotein oxidase with an extremely low kcat turnover number. Comparison of kcat/Km values suggests that PNP rather than PMP is the in vivo substrate of E. coli PdxH oxidase. In contrast, the eukaryotic enzyme has similar kcat/Km values for PNP and PMP and seems to act as a scavenger. E. coli PNP/PMP oxidase activities were competitively inhibited by the pathway end product, PLP, and by the analog, 4-deoxy-PNP, with Ki values of 8 and 105 microM, respectively. Immunoinhibition studies suggested that the catalytic domain of the enzyme may be composed of discontinuous residues on the polypeptide sequence. Two independent quantitation methods showed that PNP/PMP oxidase was present in about 700 to 1,200 dimer enzyme molecules per cell in E. coli growing exponentially in minimal medium plus glucose at 37 degrees C. Thus, E. coli PNP/PMP oxidase is an example of a relatively abundant, but catalytically sluggish, enzyme committed to PLP coenzyme biosynthesis. << Less

  J. Bacteriol. 177:883-891(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Identification of a pyridoxine (pyridoxamine) 5'-phosphate oxidase from Arabidopsis thaliana.

  Sang Y., Barbosa J.M., Wu H., Locy R.D., Singh N.K.

  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 ... >> More

  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. << Less

  FEBS Lett. 581:344-348(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Expression, purification, and characterization of recombinant Escherichia coli pyridoxine 5'-phosphate oxidase.

  di Salvo M., Yang E., Zhao G., Winkler M.E., Schirch V.

  A previously cloned pdxH gene from Escherichia coli coding for pyridoxine 5'-phosphate oxidase was transferred to a pET22b vector and expressed in E. coli HMS174(DE3) cells. The soluble overexpressed enzyme was rapidly purified in high yield using two chromatography columns with an overall purific ... >> More

  A previously cloned pdxH gene from Escherichia coli coding for pyridoxine 5'-phosphate oxidase was transferred to a pET22b vector and expressed in E. coli HMS174(DE3) cells. The soluble overexpressed enzyme was rapidly purified in high yield using two chromatography columns with an overall purification of about 2.8-fold. The purified enzyme contained tightly bound FMN. The enzyme exhibited the same spectral properties and similar kinetic constants to those previously reported by G. Zhao and M. E.Winkler (J. Bacteriol. 177, 883, 1995), but differed from the properties reported by other investigators. A rapid procedure was developed for preparing apoPNP Ox in high yield. Both the holo- and apoenzymes were homodimers. The molar absorbtivity coefficient for the protein was determined for the fully active apoPNP Ox from is amino acid composition. Using this value and the spectral properties of the bound FMN it was shown by three different methods that the dimeric enzyme contains two molecules of bound FMN per dimer and not one FMN as previously reported. << Less

  Protein Expr. Purif. 13:349-356(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Vitamer levels, stress response, enzyme activity, and gene regulation of Arabidopsis lines mutant in the pyridoxine/pyridoxamine 5'-phosphate oxidase (PDX3) and the pyridoxal kinase (SOS4) genes involved in the vitamin B6 salvage pathway.

  Gonzalez E., Danehower D., Daub M.E.

  PDX3 and SALT OVERLY SENSITIVE4 (SOS4), encoding pyridoxine/pyridoxamine 5'-phosphate oxidase and pyridoxal kinase, respectively, are the only known genes involved in the salvage pathway of pyridoxal 5'-phosphate in plants. In this study, we determined the phenotype, stress responses, vitamer leve ... >> More

  PDX3 and SALT OVERLY SENSITIVE4 (SOS4), encoding pyridoxine/pyridoxamine 5'-phosphate oxidase and pyridoxal kinase, respectively, are the only known genes involved in the salvage pathway of pyridoxal 5'-phosphate in plants. In this study, we determined the phenotype, stress responses, vitamer levels, and regulation of the vitamin B(6) pathway genes in Arabidopsis (Arabidopsis thaliana) plants mutant in PDX3 and SOS4. sos4 mutant plants showed a distinct phenotype characterized by chlorosis and reduced plant size, as well as hypersensitivity to sucrose in addition to the previously noted NaCl sensitivity. This mutant had higher levels of pyridoxine, pyridoxamine, and pyridoxal 5'-phosphate than the wild type, reflected in an increase in total vitamin B(6) observed through HPLC analysis and yeast bioassay. The sos4 mutant showed increased activity of PDX3 as well as of the B(6) de novo pathway enzyme PDX1, correlating with increased total B(6) levels. Two independent lines with T-DNA insertions in the promoter region of PDX3 (pdx3-1 and pdx3-2) had decreased PDX3 activity. Both also had decreased activity of PDX1, which correlated with lower levels of total vitamin B(6) observed using the yeast bioassay; however, no differences were noted in levels of individual vitamers by HPLC analysis. Both pdx3 mutants showed growth reduction in vitro and in vivo as well as an inability to increase growth under high light conditions. Increased expression of salvage and some of the de novo pathway genes was observed in both the pdx3 and sos4 mutants. In all mutants, increased expression was more dramatic for the salvage pathway genes. << Less

  Plant Physiol. 145:985-996(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.