Thiamine-dependent regulation of mammalian brain pyridoxal kinase in vitro and in vivo

Abstract

Vitamins B₁ (thiamine) and B₆ (pyridox (al/ine/amine)) are crucial for central nervous system (CNS) function and neurogenesis due to the coenzyme action of their phosphorylated derivatives in the brain metabolism of glucose and neurotransmitters. Here, the non-coenzyme action of thiamine on the major mammalian producers of pyridoxal-5'-phosphate (PLP), such as pyridoxal kinase (PdxK) and pyridoxine 5'-phosphate oxidase (PNPO), is characterized. Among the natural thiamine compounds, thiamine triphosphate (ThTP) is the best effector of recombinant human PdxK (hPdxK) in vitro, inhibiting hPdxK in the presence of Mg²⁺ but activating the Zn²⁺ -dependent reaction. Inhibition of hPdxK by thiamine antagonists decreases from amprolium to pyrithiamine to oxythiamine, highlighting possible dysregulation of both the B₁ - and B₆ -dependent metabolism in the chemical models of thiamine deficiency. Compared with the canonical hPdxK, the D87H and V128I variants show a twofold increase in K_app of thiamine inhibition, and the V128I and H246Q variants show a fourfold and a twofold decreased K_app of thiamine diphosphate (ThDP), respectively. Thiamine administration changes diurnal regulation of PdxK activity and phosphorylation at Ser213 and Ser285, expression of the PdxK-related circadian kinases/phosphatases in the rat brain, and electrocardiography (ECG). In contrast to PdxK, PNPO is not affected by thiamine or its derivatives, either in vitro or in vivo. Dephosphorylation of the PdxK Ser285, potentially affecting mobility of the ATP-binding loop, inversely correlates with the enzyme activity. Dephosphorylation of the PdxK Ser213, which is far away from the active site, does not correlate with the activity. The correlations analysis suggests the PdxK Ser213 to be a target of kinase MAP2K1 and phosphatase Ppp1ca. Diurnal effects of thiamine administration on the metabolically linked ThDP- and PLP-dependent enzymes may support the brain homeostatic mechanisms and physiological fitness.

Keywords: MAP kinases; Ppp1ca; diurnal rhythms; pyridoxal kinase; thiamine antagonists; thiamine triphosphate.