Pyridoxal 5′-phosphate (PLP), the catalytically active form of vitamin B6, plays a crucial biological function as enzyme cofactor, and as such has key roles in many essential metabolic pathways. PLP is a very reactive molecule, therefore its cellular concentration in the free form must be tightly regulated to avoid toxic effects. At the same time, a sufficient amount of PLP must be supplied to satisfy cell requirements. Therefore, it is expected that the encounter between PLP and apoenzymes does not take place by simple diffusion and collision, but relies on a specific transport system. Experimental evidence suggests that PLP carrier proteins exist, which bind the cofactor, protecting it from the environment and forming a PLP cellular reservoir, and transfer it to apoenzymes. While vitamin B6 metabolism has been widely investigated with respect to reaction steps and enzyme catalysis, the mechanism of PLP delivery to apoenzymes is very poorly understood. The present proposal aims to investigate the role and mechanism of action of three putative PLP-binding proteins in bacteria: YggS, pyridoxine 5’-phosphate oxidase (PNPO) and pyridoxal kinase (PDXK). Although it has been proposed that all these proteins directly participate to PLP homeostasis and delivery in the cell, their actual involvement in these processes and the molecular mechanism of PLP transfer have never been clarified. Our investigation will also be marginally devoted to human PLP carrier proteins, in particular to PNPO and the Yggs’ homologue PLP-BP (PLP-binding protein), which play essential roles in PLP homeostasis and whose mutations cause vitamin B6-dependent epilepsy. Specifically, our project will focus on: i) the mechanism of PLP transfer from PLP carrier proteins to PLP-dependent apoenzymes; ii) the protein structural features involved in PLP binding and transfer capabilities; iii) in vivo studies on the actual role of PLP binding proteins in PLP homeostasis and delivery; these studies will use Escherichia coli as bacterial model and Drosophila melanogaster as multicellular eukaryotic model. Considering the importance of vitamin B6 in bacterial physiology and virulence, the outcomes of our project may have a relevance for human health, indicating novel targets of antimicrobial intervention. Moreover, the information on the role of human PNPO and PLP-BP deriving from our studies will also be valuable, considering that imbalance of vitamin B6 homeostasis is involved in many neurological disorders.