Purification and characterization of the pyridoxol-5'-phosphate:oxygen oxidoreductase (deaminating) from Escherichia coli.
The E. coli gene pdxH encoding pyridoxol-5'-phosphate:oxygen oxidoreductase (deaminating) (EC 1.4.3.5, PdxH) was cloned, located to phage 20B5 of the library of Kohara et al. (Kohara, Y, Akiyama, K. and Isono K. (1987) Cell 50, 495-508) and assigned to a stretch between 36.0 and 36.1 min of the E. coli chromosome. The gene was overexpressed as a MBP/PdxH fusion protein. The fusion protein was purified by affinity chromatography on an amylose resin and hydrolyzed in the presence of protease 'factor Xa' resulting in homogeneous PdxH protein after another column chromatography. Both the MBP/PdxH fusion protein and the PdxH protein were characterized. Both enzymes are FMN-dependent enzymes which oxidize pyridoxol phosphate and pyridoxamine phosphate in the presence of oxygen to pyridoxal phosphate. Km values of both proteins were similar ranging from 350 to 400 microM for the two substrates. The enzymes did not accept non-phosphorylated substrates. Kinetic data indicate that the enzyme (MBP/PdxH) is product inhibited (Ki 8 microM) by pyridoxal phosphate as a mixed type inhibitor. As revealed by gel exclusion chromatography a minor fraction of the fusion protein formed a dimer, whereas the bulk amount of protein was a monomer. No indication was found that the PdxH protein forms a dimer. The monomer was shown to be catalytically active.
Notheis
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Institut für Pharmazeutische Biologie, Rheinische Friedrich-Wilhelms-Universität, Bonn, Germany.
Drewke
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Leistner
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E
D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D011732:Pyridoxal Phosphate / Q000096:biosynthesis / Q000737:chemistry; D011734:Pyridoxaminephosphate Oxidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*; D011736:Pyridoxine / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D011732:Pyridoxal Phosphate; D011734:Pyridoxaminephosphate Oxidase; D011736:Pyridoxine