Tight binding of pyridoxal 5'-phosphate to recombinant Escherichia coli pyridoxine 5'-phosphate oxidase.
Escherichia coli pyridoxine (pyridoxamine) 5'-phosphate oxidase (PNPOx) catalyzes the oxidation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate to pyridoxal 5'-phosphate (PLP) using flavin mononucleotide (FMN) as the immediate electron acceptor and oxygen as the ultimate electron acceptor. This reaction serves as the terminal step in the de novo biosynthesis of PLP in E. coli. Removal of FMN from the holoenzyme results in a catalytically inactive apoenzyme. PLP molecules bind tightly to both apo- and holoPNPOx with a stoichiometry of one PLP per monomer. The unique spectral property of apoPNPOx-bound PLP suggests a non-Schiff base linkage. HoloPNPOx with tightly bound PLP shows normal catalytic activity, suggesting that the tightly bound PLP is at a noncatalytic site. The tightly bound PLP is readily transferred to aposerine hydroxymethyltransferase in dilute phosphate buffer. However, when the PNPOx. PLP complex was added to aposerine hydroxymethyltransferase suspended in an E. coli extract the rate of reactivation of the apoenzyme was several-fold faster than when free PLP was added. This suggests that PNPOx somehow targets PLP to aposerine hydroxymethyltransferase in vivo.
Yang
E S
ES
Department of Biochemistry and Molecular Biophysics, Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Schirch
V
V
D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000378:metabolism; D012696:Glycine Hydroxymethyltransferase / Q000378:metabolism; D020035:Holoenzymes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D011734:Pyridoxaminephosphate Oxidase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013057:Spectrum Analysis
D001051:Apoenzymes; D003432:Cross-Linking Reagents; D020035:Holoenzymes; D010710:Phosphates; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D005486:Flavin Mononucleotide; D011734:Pyridoxaminephosphate Oxidase; D012696:Glycine Hydroxymethyltransferase