Titolo della tesi: Regulation of vitamin B6 metabolism in Escherichia coli
The catalytically active form of vitamin B6, pyridoxal 5'-phosphate (PLP), acts as a coenzyme in a variety of different enzymatic reactions. PLP is essential for the normal growth and development of all the organisms, but only plants and microorganisms can synthetise it de novo, while the other organisms must recycle it from nutrients.
The PLP is a very reactive molecule thanks to its aldehyde group and its intracellular concentration must be kept low to avoid undesired toxic reaction in the organism. The regulation of the free PLP concentration in cells occurs through a variety of mechanism, such as its dephosphorylation to PL or, as recently discovered, through the binding to PLP Binding Protein. In Escherichia coli an important candidate for this role has recently been discovered: YggS is a non-catalytic protein able to bind PLP. It belongs to the COG0325 family, a class of protein sharing structure homology with PLP-dependent enzymes, such as alanine racemase and some decarboxylases that have the same Fold-type III.
The metabolism of the PLP in E. coli has been studied for years, so in this work, our group have deepened the regulation of the PLP metabolism, studying the state of the art about the genes involved in PLP homeostasis and focusing the analysis on the most important genes. Our studies have analysed in vivo the phenotypes linked to the genes involved in PLP homeostasis when E. coli is grown in different media and in presence of vitamers. The study has also considered the expression of the genes involved in PLP homeostasis in presence of vitamin B6 vitamers and during different growth phases.
Finally, the attention was focused on the protein YggS, whose role has not yet been discovered, but it is hypothesised that it could be both a PLP binding protein and a carrier of this important cofactor to the apo PLP-dependent enzymes. Our studies were focused on its capacity to bind PLP and using some mutant forms of this protein, we have studied the transfer mechanism of PLP to the apo PLP-dependent enzymes.