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.
The present project stands out as an important study aimed at deeply investigating a pivotal aspect of vitamin B6 metabolism and homeostasis that has so far been neglected: the mechanism of delivery of pyridoxal 5'-phosphate to PLP-dependent enzymes. This is a poorly understood and crucial aspect of vitamin B6 metabolism that is fundamental to understand the role of this vitamin in human health and disease. As mentioned above, vitamin B6 and vitamin B6-dependent enzymes play pivotal roles in many human disorders and the outcomes of our studies will help understanding the actual role of PLP-BP in human vitamin B6 metabolism. Therefore, although we will tackle basic science questions, the results obtained in our project may have important medical implications.
The scientific, technological and social/economic impact of the project is the following.
Scientific impact. The increased scientific knowledge based on original findings is expected to be the main outcome of the present project. In particular, we will: (i) clarify the mechanism of PLP transfer from PLP-BP to the enzymes that use it as cofactor; ii) identify the molecular basis of vitamin B6-dependent epilepsy; iii) characterize the structural features of PLP-BP involved in PLP transfer; iv) test our findings and hypothesis in vivo using E. coli as model. The outcome of our investigations will help understanding human vitamin B6 metabolism better.
Technological impact. All methodologies that will be set up and developed in the project will be available in the future to study PLP transfer from other PLP-carrier proteins such as PNPOx and PLK, which are also of crucial importance in vitamin B6 metabolism and are involved in severe human diseases.
Social/economic impact. Vitamin B6 metabolism plays a crucial role in human health and disease. As mentioned above, several important neurological diseases are connected to perturbations of PLP homeostasis. A better understanding of the role played by PLP-BP in this aspect of human metabolism may have future medical implications.