Pyridoxal-5-phosphate (PLP) dependent enzymes are a large and catalytically diversegroup of proteins primarily involved in the metabolism of amino acids, amino acid derived compounds, and amino sugars. In this work, three PLP-dependent enzymes, Homo sapiens kynureninase, Pseudomonas dacunhae L-aspartate-2-decarboxylase, and Pyrococcus furiosustryptophan synthase -subunit 2homolog are studied using a variety of biophysical methods. Thenovel crystal structures for these enzymes are presented, along with the structure of a kynureninase-inhibitor complex, and the analysis of a number of mutants generated to study specific structure activity relationships in them. The results of these analyses reveal the interactions that contribute to substrate specificity in kynureninase, a novel oligomerization scheme and catalytically important residues in aspartate-2-decarboxylase, and the elucidation of the kinetic properties of the P. furiosus tryptophan synthase 2-subunit homolog.