Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, plays a crucial biological function as enzyme cofactor, and as such has key roles in many essential metabolic pathways. Humans are not able to synthesise PLP, but recycle it through a salvage pathway that interconverts B6 vitamers present in food. PLP is a very reactive molecule, therefore its cellular concentration in the free form must be tightly regulated to avoid toxicity. At the same time, sufficient amounts of PLP must be present to satisfy cell requirements. It follows that the encounter between PLP and apoenzymes cannot take place by simple collision, but has to rely on specific transport systems. PLP carrier proteins must exist that bind the cofactor, protecting it from the environment, and transfer it to apoenzymes. While vitamin B6 metabolism has been widely investigated with respect to reaction mechanisms and catalysis, PLP delivery systems are very poorly understood, although they are involved in severe human disorders. The present proposal aims to investigate the role and mechanism of action of the human PLP-binding protein (PLP-BP), which plays an essential role in PLP homeostasis, since its mutations cause vitamin B6-dependent epilepsy. Although it has been proposed that PLP-BP directly participates to PLP delivery in the cell, its actual capability to transfer PLP to apoenzymes has never been demonstrated. Moreover, the molecular basis of the pathogenic effect caused by PLP-BP mutations are unknown. We intend to investigate the mechanism of PLP transfer from human PLP-BP to human PLP-dependent apoenzymes. We will also analyse the protein structural features involved in this process, with particular focus on pathogenic PLP-BP mutants. Finally, in vivo studies will be carried out on an E. coli strain whose homologous PLP-BP has been knocked out. Expression of wild type and mutant PLP-BP forms in this strain will allow complementation and biochemical characterisation studies.
