Pyridoxal phosphate (PLP)-independent racemases, enzymes that are cofactor and metal free invert, the configuration at the α-carbon of amino acids. In nature, diaminopimelate epimerase (DapF), glutamate racemase (GluR), aspartate racemase (AspR), proline racemase (ProR), O-ureidoserine racemase (DcsC), and a putative amino acid racemase from Bacillus (RacX) are known as PLP-independent racemases. Comparison of these enzymes, may reveal similarities and differences in their unusual mechanism of action. The first part of this dissertation describes mechanistic studies of O-ureidoserine racemase (DcsC). DcsC is a PLP-independent enzyme that is involved in the biosynthetic route of the antibiotic D-cycloserine. To obtain insight into the mechanism of DcsC, a variant featuring an N-terminal SUMO-tag was expressed possessing significantly enhanced stability and activity. Synthesis of enantiomerically pure inhibitors in combination with site-specific mutation of active site cysteines to serines of this enzyme offers information about the mechanism of this transformation. Homology modeling with a close relative (diaminopimelate epimerase, DapF) inspired C- and N-terminal truncation of DcsC to produce a more compact yet still active enzyme variant. An ongoing goal of this project is obtaining a single X-ray structure of the enzyme to understand the interior active site dimensions and recognition of the distal site. The second part of this thesis describes efforts on the characterization of another amino acid racemase, namely RacX. RacX is also a PLP-independent enzyme that is found almost exclusively in Bacillus species. Even though the enzyme is highly homologous to AspR and GluR, aspartate and glutamate are not good substrate for RacX. Since the catalytic efficiency for other amino acids with RacX was also low, it is possible that the natural substrate of RacX is not a single amino acid but a derivative thereof. The RacX gene is clustered with penicillin binding protein 4 (PbpE) which catalyzes the process of peptidoglycan hydrolysis. Therefore, it may be that RacX has a substrate which is a part of the peptidoglycan structure. To investigate this, various oligopeptides were synthesized corresponding to peptides in peptidoglycan. Further synthesis of proposed substrates and testing of their catalytic transformation are ongoing to determine if they can be efficiently transformed.