Vitamin B1 is essential for all organisms being well recognized as a necessary cofactor for key metabolic pathways such as glycolysis and was more recently implicated in DNA damage responses. Little is known about the enzyme responsible for the formation of the pyrimidine moiety (HMP-P synthase). We report a structure-function study of the HMP-P synthase from yeast, THI5p. Our crystallographic structure shows that the THI5p is a mix between periplasmic binding proteins and pyridoxal 5'-phosphate (PLP) dependent enzymes. Mutational and yeast complementation studies identify the key residues for HMP-P biosynthesis and offers unprecedented insights into the use of PLP as a substrate rather than as a cofactor, the requirement of iron and more generally into the HMP-P synthase reaction mechanism.