Molecular Basis of Reduced Pyridoxine 5′-Phosphate Oxidase Catalytic Activity in Neonatal Epileptic Encephalopathy Disorder

Mutations in pyridoxine 5′-phosphate oxidase are known to cause neonatal epileptic encephalopathy. This disorder has no cure or effective treatment and is often fatal. Pyridoxine 5′-phosphate oxidase catalyzes the oxidation of pyridoxine 5′-phosphate to pyridoxal 5′-phosphate, the active cofactor fo...

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Bibliographic Details
Published in:The Journal of biological chemistry 2009-11, Vol.284 (45), p.30949-30956
Main Authors: Musayev, Faik N., Di Salvo, Martino L., Saavedra, Mario A., Contestabile, Roberto, Ghatge, Mohini S., Haynes, Alexina, Schirch, Verne, Safo, Martin K.
Format: Article
Language:English
Subjects:
Binding Sites
Catalysis
Crystallization
Epilepsy, Benign Neonatal - enzymology
Epilepsy, Benign Neonatal - genetics
Flavin Mononucleotide - chemistry
Humans
Kinetics
Molecular Conformation
Mutation, Missense
Protein Binding
Protein Structure and Folding
Pyridoxal Phosphate - analogs & derivatives
Pyridoxal Phosphate - chemistry
Pyridoxaminephosphate Oxidase - chemistry
Pyridoxaminephosphate Oxidase - genetics
Pyridoxaminephosphate Oxidase - metabolism
Substrate Specificity
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Summary:Mutations in pyridoxine 5′-phosphate oxidase are known to cause neonatal epileptic encephalopathy. This disorder has no cure or effective treatment and is often fatal. Pyridoxine 5′-phosphate oxidase catalyzes the oxidation of pyridoxine 5′-phosphate to pyridoxal 5′-phosphate, the active cofactor form of vitamin B6 required by more than 140 different catalytic activities, including enzymes involved in amino acid metabolism and biosynthesis of neurotransmitters. Our aim is to elucidate the mechanism by which a homozygous missense mutation (R229W) in the oxidase, linked to neonatal epileptic encephalopathy, leads to reduced oxidase activity. The R229W variant is ∼850-fold less efficient than the wild-type enzyme due to an ∼192-fold decrease in pyridoxine 5′-phosphate affinity and an ∼4.5-fold decrease in catalytic activity. There is also an ∼50-fold reduction in the affinity of the R229W variant for the FMN cofactor. A 2.5 Å crystal structure of the R229W variant shows that the substitution of Arg-229 at the FMN binding site has led to a loss of hydrogen-bond and/or salt-bridge interactions between FMN and Arg-229 and Ser-175. Additionally, the mutation has led to an alteration of the configuration of a β-strand-loop-β-strand structure at the active site, resulting in loss of two critical hydrogen-bond interactions involving residues His-227 and Arg-225, which are important for substrate binding and orientation for catalysis. These results provide a molecular basis for the phenotype associated with the R229W mutation, as well as providing a foundation for understanding the pathophysiological consequences of pyridoxine 5′-phosphate oxidase mutations.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M109.038372