Molecular characterization of pyridoxine 5′-phosphate oxidase and its pathogenic forms associated with neonatal epileptic encephalopathy

Abstract

Defects of vitamin B 6 metabolism are responsible for severe neurological disorders, such as pyridoxamine 5′-phosphate oxidase deficiency (PNPOD; OMIM: 610090), an autosomal recessive inborn error of metabolism that usually manifests with neonatal-onset severe seizures and subsequent encephalopathy. At present, 27 pathogenic mutations of the gene encoding human PNPO are known, 13 of which are homozygous missense mutations; however, only 3 of them have been characterised with respect to the molecular and functional properties of the variant enzyme forms. Moreover, studies on wild type and variant human PNPOs have so far largely ignored the regulation properties of this enzyme. Here, we present a detailed characterisation of the inhibition mechanism of PNPO by pyridoxal 5′-phosphate (PLP), the reaction product of the enzyme. Our study reveals that human PNPO has an allosteric PLP binding site that plays a crucial role in the enzyme regulation and therefore in the regulation of vitamin B 6 metabolism in humans. Furthermore, we have produced, recombinantly expressed and characterised several PNPO pathogenic variants responsible for PNPOD (G118R, R141C, R225H, R116Q/R225H, and X262Q). Such replacements mainly affect the catalytic activity of PNPO and binding of the enzyme substrate and FMN cofactor, leaving the allosteric properties unaltered.

Identification and characterization of the pyridoxal 5’-phosphate allosteric site in Escherichia coli pyridoxine 5’-phosphate oxidase

“…On the other hand, PLP binding at the active site is positively influenced by PLP binding at the allosteric site (and this is a reciprocal effect), as indicated by a factor γ = 0.31. Of interest, we observed a similar behavior with human PNPO ( 6 ) ( Fig. 2 B ), although in this case γ was equal to 6.6.…”

“…The PEP ternary complex can be formed, but only upon binding of PLP to the active site of the PE complex. In this respect, this model is similar to that used to explain the PLP inhibition behavior of the human pyridoxine 5’-phosphate oxidase ( 6 ). C , also, in this model some of the binding equilibria present in the general inhibition model ( A ) do not take place and are therefore shown in red .…”

“…The The quadruple mutant conserves the structural elements of the wild type protein: 5 alpha helices, numbered from H1 to H5, and 10 β-strands. In addition, in the PLP-free ePNPOqm the N-terminal region is also visible, folded in an additional α-helix (H0, residues [5][6][7][8][9][10][11][12][13][14][15][16][17][18].…”

“…PLP allosteric inhibition is also observed with human PNPO (6). However, in this case the coupling between the allosteric site and the active site is weaker, so that the PES complex maintains a partial catalytic activity and PLP can also bind at the active site, forming an enzyme complex with two PLP molecules (PEP in Scheme 1B).…”

“…The PES ternary complex is catalytically inactive. B , rapid equilibrium kinetics model corresponding to the parabolic inhibition observed with human pyridoxine 5’-phosphate oxidase ( 6 ). This model is similar to that shown in A ; however, in this case, the allosteric coupling between the allosteric site and the active site is weaker (the α value is about 3) and the PES ternary complex is catalytically active, although k CAT is reduced by a β coefficient (equal to 0.15).…”