Comprehension of two proteins involved in vitamin B6-responsive seizures, in light of the metabolite repair concept

Details

Supervisors

Van Schaftingen, Emile ; Marbaix, Alexandre

Faculty

Faculté de médecine et médecine dentaire

Degree label

Master [180] en médecine

Abstract

Pyridoxal 5’-phosphate (PLP), the active form of vitamin B6, is a cofactor for more than 60 enzymes in humans mainly involved in amino acids and neurotransmitters metabolism. Thus, defects in PLP metabolism may lead to multiple disorders, mainly seizures that are refractory to common anticonvulsants but respond very well to pyridoxal-phosphate (PLP) and/or pyridoxine (PN). Multiple inborn errors affecting vitamin B6 metabolism have been described so far, such as pyridox(am)ine phosphate oxidase (PNPO) deficiency and PROSC/PLPBP deficiency. In this work, following an approach based on the metabolite repair concept, we discovered a new enzymatic activity of PNPO and we tried to elucidate the function of PROSC. Concerning PNPO, we discovered that the protein catalyses the conversion of 6NADH and 6NADPH to NAD+ and NADP+, respectively, contributing to the repair of these abnormal nucleotides along with renalase (RNLS). 6NADH and 6NADPH are isomers of the classical pyridine nucleotides NADH and NADPH, and are strong inhibitors of several dehydrogenases. While an FAD-linked enzyme called renalase is known to catalyse 6NAD(P)H oxidation, we have several experimental data supporting the idea that pyridox(am)ine phosphate oxidase contributes importantly in this repair system. In addition, we tried to identify the critical residues for the 6NADH oxidase activity in N. punctiforme PPOX2, a PNPO-related protein present in a few species including plants, yeasts and cyanobacteria, which show ≈ 10% identity with bona fide PNPOs and acts very well on 6NADH and 6NADPH, but is inactive on pyridox(am)ine-phosphate. All PPOX2 mutants showed a decrease in the 6NADH oxidase activity and in their affinity for 6NAD(P)H. This was especially striking with the mutant R45A, where the arginine 45 is replaced with an alanine, supporting the prominent role of this residue. PROSC belongs to a ubiquitously distributed family of PLP binding proteins which is apparently involved in PLP homeostasis, though its exact function is unknown. We tested if thiazolidine or oxazolidine complexes of PLP were metabolised by PROSC, but no activity was observed. In parallel, liquid chromatography-mass spectrometry (LC-MS) analyses on YggS (the E. coli homolog of PROSC) deficient cells showed an accumulation of pyridoxine- phosphate (PNP) and of another compound that may correspond to a PLP isomer or a molecule containing a PLP moiety, which would be fragmented during MS analysis. It is tempting to speculate that this compound is normally metabolized by YggS and that its accumulation in the absence of YggS perturbs the function and the metabolism of PLP.