Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, plays a crucial role in many essential metabolic pathways. PLP is a very reactive molecule, therefore its cellular concentration in the free form must be low to avoid toxic effects. At the same time, large amounts of PLP must be available to satisfy cell needs. How these requirements are met is an obscure aspect of vitamin B6 metabolism. Pyridoxine 5'-phosphate (PNPO) is a crucial enzyme in PLP biosynthesis and recycling in bacteria and humans, since it catalyses PLP formation from B6 vitamers precursors. In particular, in humans, mutations of PNPO are responsible of severe neurological disorders. It is known that, in vitro, PLP tightly binds to PNPO inhibiting its catalytic activity; in vitro, this tightly bound PLP can be transferred to apoenzymes that require it as cofactor. However, the molecular mechanisms underlying these processes are unknown. Moreover, the actual importance of such features of PNPO, observed in vitro, in PLP homeostasis and delivery in the cell is unknown. We have recently uncovered that PLP acts on E. coli and human PNPO through an allosteric feedback inhibition mechanism. We have also identified the location of the allosteric site in the structure of E. coli PNPO. The main aim of our project is to unravel the actual role of the allosteric PNPO binding site in PLP homeostasis and delivery to apoenzymes. In order to achieve this goal, we will: i) produce and characterise mutant forms of human and E. coli PNPO in which either the allosteric or the catalytic properties of the enzyme have been abolished; ii) use these PNPO mutant forms in complementation experiments that will use E. coli as model system to understand the role the PNPO allosteric site in vivo. Considering the importance of vitamin B6 in bacterial virulence and human health, the outcomes of our project may have a medical relevance.
