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. 2009 Nov 6;284(45):30949-56.
doi: 10.1074/jbc.M109.038372. Epub 2009 Sep 15.

Molecular basis of reduced pyridoxine 5'-phosphate oxidase catalytic activity in neonatal epileptic encephalopathy disorder

Faik N Musayev  1 , Martino L Di Salvo, Mario A Saavedra, Roberto Contestabile, Mohini S Ghatge, Alexina Haynes, Verne Schirch, Martin K Safo
Affiliations

Molecular basis of reduced pyridoxine 5'-phosphate oxidase catalytic activity in neonatal epileptic encephalopathy disorder

Faik N Musayev et al. J Biol Chem. .

Abstract

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 B(6) 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 approximately 850-fold less efficient than the wild-type enzyme due to an approximately 192-fold decrease in pyridoxine 5'-phosphate affinity and an approximately 4.5-fold decrease in catalytic activity. There is also an approximately 50-fold reduction in the affinity of the R229W variant for the FMN cofactor. A 2.5 A 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 beta-strand-loop-beta-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.

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Figures

FIGURE 1.
FIGURE 1.
Fluorescence emission spectra of FMN (excitation wavelength of 450 nm) upon binding to apo forms of PNP oxidase. Continuous line, wild type; dashed line, R229Q; dotted line, R229W.
FIGURE 2.
FIGURE 2.
Structure of PNP oxidase enzyme. All figures were drawn using PyMOL (45). A, least-squares superposition of human PNP oxidase R229W dimeric structure in complex with FMN and PLP (red) with that of human PNP oxidase wild-type dimeric structure in complex with FMN and PLP (blue). B, a difference electron density map (with coefficients Fo − Fc shown at the 2.2 σ level) of the R229W model calculated before FMN, PLP, and the Trp-229 side chain were added to the model. C, an electron-density map (with coefficients 2Fo − Fc shown at the 0.8 σ level) of the active site of the refined R229W model. All maps are superimposed with the final refined models. D, stereo view comparison of the FMN phosphate (Phosph) binding site (residue 229 environment) of the wild-type structure (blue) and R229W structure (red). Residue 229 is labeled as X229. E, stereo view comparison of the active site of the wild-type structure (blue) and R229W structure (red). Note the significant movements of the R229W mutant residues His-227 and Arg-225 away from the wild-type position.
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