Kinetic limitation and cellular amount of pyridoxine (pyridoxamine) 5'-phosphate oxidase of Escherichia coli K-12.
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.
Department of Microbiology and Molecular Genetics, University of Texas, Houston Medical School 77030.
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Winkler
D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000032:analysis; D005420:Flavoproteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D011734:Pyridoxaminephosphate Oxidase / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism
D005420:Flavoproteins; D011994:Recombinant Proteins; D005486:Flavin Mononucleotide; D006861:Hydrogen Peroxide; D011734:Pyridoxaminephosphate Oxidase