The metabolic network within organisms is comprised of individual modules that are interconnected. Although the overall architecture of individual networks can be predicted due to the advent of affordable whole genome sequencing, subtle differences in network structure lead to differences in function of individual modules between organisms. In addition, moving metabolic modules between organisms in an effort to build new pathways is encumbered by the lack of knowledge about the functional relationships within and between metabolic networks. Thus, metabolic engineering instead relies on building heterologous pathways by tedious trial and error. Thiamine biosynthesis has been used as a tool to dissect connections within metabolic networks and can be used to extend our understanding about the functional relationships found within metabolism. This thesis uses Thi5 as a model metabolic node to understand the differences between the metabolic networks of Saccharomyces cerevisiae, Legionella pneumophila and Salmonella enterica and determine the new metabolic requirements for 4-amino-5- hydroxymethyl-2-methylpyrimidine-phosphate (HMP-P) biosynthesis when Thi5 is incorporated into the metabolic network of S. enterica. The first part of this work identifies a requirement of additional pyridoxal-5’-phosphate (PLP) synthases for ScThi5 function in the native network of S. cerevisiae. The second investigation extends the Thi5-dependent pathway for HMP-P biosynthesis into Legionella pneumophila and identified functional differences between ScThi5 and LpThi5. Finally, the third investigation identifies that both alpha-ketoglutarate and PLP are required to integrate ScThi5 into the S. enterica metabolic network and suggests that structural differences between LpThi5 and ScThi5 may explain differences in function.