One of the major and underappreciated issues of the post-genomic era is that the precise molecular functions of half of the proteins in databases are not known. Even in Escherichia coli K12 MG1655 one of the most studied model organism, ~1200 genes are still of unknown (or erroneous) function. In parallel, open questions remain in metabolic areas that were thought to be well studied like vitamin synthesis and salvage. For example, vitamin B6 in its active form as pyridoxal 5'-phosphate (PLP) is a cofactor for over 180 PLP-dependent enzymes that are involved in many metabolic pathways. PLP is a highly reactive molecule and is both labile and toxic so the intracellular concentration of PLP not bound to proteins remains low. How PLP is delivered to target enzymes in these conditions remains a mystery in all organisms. In addition, while the core E. coli B6 synthesis pathway is well described, the transporter(s) involved in salvage are unidentified and very little is known about regulation of PLP synthesis/salvage genes. This project focuses on identifying “missing” players in PLP metabolism in E. coli. In this process, the functions of several orphan E. coli genes should be solved and, more generally, key conserved players in PLP homeostasis characterized. Preliminary results published in two manuscripts have shown that the E. coli YggS protein, a member of the COG0325 as well as its human ortholog PROSC that has been linked to PLP-dependent diseases are involved in PLP homeostasis. The goal of Aim 1 is to test the hypothesis that this family could be the “missing” key player in delivering the PLP cofactor to target enzymes. Aim 2 focuses the response to PLP imbalance by identifying missing E. coli B6 transporters and characterizing the response to high Pyridoxin levels. The final exploratory aim will focus on two other orphan PLP binding proteins and an orphan PLP-binding regulator. The proposed research is significant because in humans, toxic levels of PLP or deficiency of PLP or defects affecting PLP metabolism are linked to many pathologies, particularly of the nervous system, hence understanding how PLP is delivered to target enzymes will have implications for a wide range of human diseases. Also, many enzymes of PLP metabolism have been recognized as antibacterial and antiparasitic targets, thus understanding PLP homeostasis is critical to develop successful inhibitors. Finally, this work should characterize 5 genes of unknown functions in E. coli, a major model organism.