The catalytically active form of vitamin B6, pyridoxal 5'-phosphate (PLP), acts as a cofactor in over 160 enzyme reactions. The cellular PLP concentration is finely regulated, since too high or too low levels of this cofactor may result in severe cellular dysfunctions. However, how PLP homeostasis is achieved is poorly understood. Studies on a ubiquitous protein, member of the COG0325 family, have recently highlighted its involvement in the control of vitamin B6 metabolism. This protein, previously named YggS in E. coli and PROSC in humans, has now been named PLP Homeostasis Protein (PLPHP), although its mechanism of action in PLP homeostasis is obscure. The three-dimensional structure of PLPHP is known and it is clear that the protein binds PLP through a Schiff base linkage with an active site lysine residue. Several hypothesis concerning PLPHP function have been proposed, among which its action as a carrier protein that is able to transfer PLP to the apoenzymes that use it as cofactor through a direct channelling mechanism.
We have recently expressed and purified the E. coli homologue of PLPHP, which is called YggS, and characterised it with respect to PLP binding and transfer properties. We have not found any evidence of PLP channelling. However, we have serendipitously discovered that, when the active site lysine residue K36 is replaced with an alanine residue, the protein is still able to bind PLP with good affinity. This unusual observation indicates that lysine residues other than K36 may be involved in the protein function, and we have preliminary data suggesting that this may be the case. In particular we believe that such lysine residues may be involved in the conformational change observed upon PLP binding. The aim of the present project is to carry out in vitro and in vivo studies to define the role of these lysine residues in the structure and function of YggS, and thereby look for a clue to the protein's role in PLP homeostasis.
The mechanisms underlying PLP homeostasis are largely unknown at present; however their understanding is of considerable importance given the crucial role of PLP metabolism in bacteria and humans. PLP has an important role in bacterial life and virulence. As mentioned above, mutations affecting the human gene encoding PLPHP cause a rare but severe form of epilepsy. PLPHP surely plays a pivotal role in PLP homeostasis in all organisms, but its mechanism of action is unknown. The present project stands out as an important study aimed at deeply investigating the structural and functional features of Escherichia coli PLPHP. A part of the project will be devoted to in vivo studies on Drosophila PLPHP, which is homologous to human PLPHP. The outcomes of our studies will help understanding the actual role of PLPHP in PLP homeostasis. Therefore, although we will tackle basic science questions, the results obtained in our project may have important scientific and 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.
Technological impact. All methodologies that will be set up and developed in the project will be available in the future to study the structural and functional features of human PLPHP, which is one of our future goals.
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 PLPHP in this aspect of metabolism may have future medical implications.