Pyridoxal Kinase: Its Role in Vitamin B6 Metabolism

Pyridoxal kinase (PL kinase) and pyridoxine 5’-phosphate oxidase (PNP oxidase) are the two vitamin B6 salvage enzymes involved in metabolism of the primary inactive vitamin B6 (pyridoxal, pyridoxine and pyridoxamine) into the active cofactor form, pyridoxal 5’-phosphate (PLP). PLP, arguably the most important vitamin, is required by numerous vitamin B6 (PLP-dependent) enzymes as a co-factor. These enzymes serve vital roles in the metabolism of glucose, lipids, amino acids, heme, DNA/RNA and many neurotransmitters. High levels of vitamin B6 are linked to neurotoxicity, due to the non-specific interactions of PLP with non-B6 proteins. This problem is controlled, in part, by maintaining a low in vivo concentration of free PLP (~1 μM); raising the intriguing question of how the cell regulates, as well as, supplies sufficient PLP to meet the requirements of B6 enzymes. Similar to PLP excess, PLP deficiency, due to mutations in PL kinase and PNP oxidase or drug-induced inhibition of their activity, has been implicated in many pathological conditions. The objective of this study is to elucidate the mechanisms underlying PLP regulation by PL kinase, and its subsequent transfer to dozens of PLP-dependent enzymes. A second objective is to gain valuable information into whether a missense mutation (S261F) in PL kinase could affect the enzyme activity and/or structure. A third objective is to understand how vitamin B6 metabolism by PL kinase is disrupted by the neurotoxic compound, ginkgotoxin. The mutant (hPL kinase S261F) was obtained using site-directed mutagenesis. It was then expressed, purified and analyzed by circular dichroism, fluorescence spectroscopy, enzyme kinetics and native-PAGE. Our results showed no considerable differences between wild-type enzyme and the mutant, suggesting the mutation to be non-pathogenic. PLP was found to inhibit PL kinase by binding to the substrate PL site in the presence of substrate MgATP to form an abortive ternary complex (PL kinase-PLP-MgATP). The physiological significance of this ternary complex was also analyzed and it was found to be a source of PLP transfer to apo B6 enzymes. Enzyme kinetics, affinity chromatography and fluorescence polarization techniques were used to test our hypothesis that the reactive PLP is transferred from PL kinase to apo-B6 enzymes via channeling. Channeling should provide an efficient and protected way for PLP transfer from the kinase or oxidase to apo-B6 enzymes. Our results provide a strong support to the channeling mechanism. Ginkgotoxin was found to be a competitive inhibitor of PL kinase with a Ki of 18 μM. X-ray crystallographic analysis of its binding mode to PL kinase confirmed its binding to the substrate PL site of the enzyme. A unique hydrophobic interaction between its lipophilic side chain 4’-OCH3 and nearby Tyr127 and Val231, in addition to the conserved PL binding interactions, was found to be responsible for its higher affinity to the enzyme