Abstract
Several variants of the enzyme pyridox(am)ine 5′-phosphate oxidase (PNPO), responsible for a rare form of vitamin B6-dependent neonatal epileptic encephalopathy known as PNPO deficiency (PNPOD), have been reported. However, only a few of them have been characterised with respect to their structural and functional properties, despite the fact that the knowledge of how variants affect the enzyme may clarify the disease mechanism and improve treatment. Here, we report the characterisation of the catalytic, allosteric and structural properties of recombinantly expressed D33V, R161C, P213S, and E50K variants, among which D33V (present in approximately 10% of affected patients) is one of the more common variants responsible for PNPOD. The D33V and E50K variants have only mildly altered catalytic properties. In particular, the E50K variant, given that it has been found on the same chromosome with other known pathogenic variants, may be considered non-pathogenic. The P213S variant has lower thermal stability and reduced capability to bind the FMN cofactor. The variant involving Arg161 (R161C) largely decreases the affinity for the pyridoxine 5′-phosphate substrate and completely abolishes the allosteric feedback inhibition exerted by the pyridoxal 5′-phosphate product.
Highlights
Istituto di Biologia e Patologia Molecolari, Consiglio Nazionale delle Ricerche, 00185 Rome, Italy; Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Sapienza Università di Roma, 00185 Rome, Italy; Genetics and Genomic Medicine, UCL Great Ormond Street Institute of Child Health, Belozersky Institute of Physico-Chemical Biology, Faculty of Bioengineering and Bioinformatics, Department of Biochemistry, Sechenov University, 119991 Moscow, Russia
Pyridox(am)ine 50 -phosphate oxidase deficiency (PNPOD; OMIM: 610090) is an autosomal recessive inborn error of metabolism caused by mutations in the gene encoding pyridox(am)ine 50 -phosphate oxidase (PNPO)
The pyridox(am)ine -phosphate oxidase (PNPO) variants D33V, E50K, R161C, and P213S were produced and purified following the protocol used for the wild type enzyme (Materials and Methods)
Summary
1. Introduction with regard to jurisdictional claims in Pyridox(am)ine 50 -phosphate oxidase deficiency (PNPOD; OMIM: 610090) is an autosomal recessive inborn error of metabolism caused by mutations in the gene encoding pyridox(am)ine 50 -phosphate oxidase (PNPO). Introduction with regard to jurisdictional claims in Pyridox(am)ine 50 -phosphate oxidase deficiency (PNPOD; OMIM: 610090) is an autosomal recessive inborn error of metabolism caused by mutations in the gene encoding pyridox(am)ine 50 -phosphate oxidase (PNPO) Patients with this disorder normally present in the neonatal period with seizures that are refractory to conventional anticonvulsant treatments but respond to treatment with supraphysiological doses of pyridoxal 50 -phospate (PLP). PNPO is a fundamental FMN-dependent enzyme that produces PLP by oxidizing pyridoxine 50 -phosphate (PNP) and pyridoxamine 50 -phosphate (PMP) recycled from non-pyridoxal B6 vitamers in the diet and generated by metabolism and protein turnover [2]. PLP, the catalytically active form of vitamin B6 , is the cofactor for about 4% of all known cellular enzymes [3], many of which are involved in the biosynthesis and metabolism of neurotransmitters and neuromodulators in the brain, including dopamine, γ-aminobutyric acid (GABA), serotonin, histamine, D-serine, and published maps and institutional affiliations
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