Maintenance of cellular vitamin B6 levels and mitochondrial oxidative function depend on pyridoxal 5′-phosphate homeostasis protein
Jolita Čiapaitė, Carlo W.T. van Roermund, Marjolein Bosma, Johan Gerrits, Sander M. Houten, Lodewijk IJlst, Hans R. Waterham, Clara van Karnebeek, Ronald J. A. Wanders, Fried Zwartkruis, Judith Jans, Nanda M. Verhoeven‐Duif
Abstract
Recently, biallelic variants in PLPBP coding for pyridoxal 5'-phosphate homeostasis protein (PLPHP) were identified as a novel cause of early-onset vitamin B 6 -dependent epilepsy. The molecular function and precise role of PLPHP in vitamin B 6 metabolism are not well understood. To address these questions we used PLPHP deficient patient skin fibroblasts and HEK293 cells, and YBL036C (PLPHP ortholog) deficient yeast. We showed that independent of extracellular B 6 vitamer type (pyridoxine, pyridoxamine or pyridoxal), intracellular PLP was lower in PLPHP deficient fibroblasts and HEK293 cells compared to controls. Culturing cells with pyridoxine or pyridoxamine led to the concentration-dependent accumulation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate (PMP), respectively, suggesting insufficient pyridox(am)ine 5′-phosphate oxidase (PNPO) activity. Experiments utilizing 13 C 4 -pyridoxine confirmed lower PNPO activity and revealed increased fractional turnovers of PLP and pyridoxal, indicating increased PLP hydrolysis to pyridoxal in PLPHP deficient cells. This effect could be partly counteracted by inactivation of pyridoxal phosphatase. PLPHP deficiency had a distinct effect on mitochondrial PLP and PMP, suggesting impaired activity of mitochondrial transaminases. Moreover, in YBL036C deficient yeast PLP was depleted and PMP accumulated only with carbon sources requiring mitochondrial metabolism. Lactate and pyruvate accumulation along with the decrease of tricarboxylic acid cycle intermediates downstream of α-ketoglutarate suggested impaired mitochondrial oxidative metabolism in PLPHP deficient HEK293 cells. We hypothesize that impaired activity of mitochondrial transaminases may contribute to this depletion. Taken together, our study provides new insights into the pathomechanisms of PLPBP deficiency and reinforces the link between PLPHP function, vitamin B 6 metabolism and mitochondrial oxidative metabolism.