1JNW

Active Site Structure of E. coli pyridoxine 5'-phosphate Oxidase

Summary for 1JNW

• Entry DOI: 10.2210/pdb1jnw/pdb
• Related: 1dnl 1g76 1g77 1g78 1g79
• Descriptor: Pyridoxine 5'-phosphate oxidase, PHOSPHATE ION, FLAVIN MONONUCLEOTIDE, ... (5 entities in total)
• Functional Keywords: n-terminal segment, plp, fmn, oxidoreductase
• Biological source: Escherichia coli
• Total number of polymer chains: 1
• Total formula weight: 26618.12

Authors

di Salvo, M.L.,Ko, T.P.,Musayev, F.N.,Raboni, S.,Schirch, V.,Safo, M.K. (deposition date: 2001-07-25, release date: 2001-08-01, Last modification date: 2023-11-15)

Primary citation

di Salvo, M.L.,Ko, T.P.,Musayev, F.N.,Raboni, S.,Schirch, V.,Safo, M.K.
Active site structure and stereospecificity of Escherichia coli pyridoxine-5'-phosphate oxidase.
J.Mol.Biol., 315:385-397, 2002

PubMed Abstract: 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.

PubMed: 11786019
DOI: 10.1006/jmbi.2001.5254

Experimental method

X-RAY DIFFRACTION (2.07 Å)