1jnw
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Active Site Structure of E. coli pyridoxine 5'-phosphate Oxidase
OverviewOverview
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.
About this StructureAbout this Structure
1JNW is a Single protein structure of sequence from Escherichia coli with , and as ligands. Active as Pyridoxal 5'-phosphate synthase, with EC number 1.4.3.5 Full crystallographic information is available from OCA.
ReferenceReference
Active site structure and stereospecificity of Escherichia coli pyridoxine-5'-phosphate oxidase., di Salvo ML, Ko TP, Musayev FN, Raboni S, Schirch V, Safo MK, J Mol Biol. 2002 Jan 18;315(3):385-97. PMID:11786019
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