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.
