Dissertation (Ph.D.)--Auburn University, 2009. / Abstract. Includes bibliographic references (p.142-154).

Thesis(M.S.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references (p.90-94).

In 2007, a gene homolog of CDO encoded by the gene Gm237 in the DUF164 family was identified as cysteamine dioxygenase (ADO). ADO is one of the only known thiol dioxygenases found in mammals. Both ADO and its partner cysteine dioxygenase (CDO) are non-heme iron dependent enzymes that play a crucial role in the biosynthesis of taruine/hypotaurine by insertion of a dioxygen molecule. However, ADO has been overshadowed by CDO as heavy research focus on CDO over the past decade has led to the elucidation of its structure and possible mechanistic properties. In an effort to further understand ADO’s mechanism and regulating role in vivo, this work will be focused on the mammalian hADO and trying to gain further insight on hADO’s structural features via crystallography work. Investigation of the crystallization parameters for hADO has elucidated several potential conditions. Detailed work on these crystallization parameters will be presented.

The function of the metalloenzymes is mainly determined by four structural features: the metal core, the metal binding motif, the second sphere residues in the active site and the electronic statistics. Cysteamine dioxygenase (ADO) and cysteine dioxygenase (CDO) are the only known enzymes that oxidize free thiol containing molecules in mammals by inserting of a dioxygen molecue. Both ADO and CDO are known as non-heme iron dependent enzymes with 3-His metal binding motif. However, the mechanistic understanding of both enzymes is obscure. The understanding of the mechanistic features of the two thiol dioxygenases is approached through spectroscopic and metal substitution in this dissertation. Another focus of the dissertation is the understanding of the function of a second sphere residue His228 in a 3-His-1-carboxyl zinc binding decarboxylase α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD). ACMSD catalyzes the decarboxylation through a hydrolase-like mechanism that is initialized by the deprotonation of metal bounded water molecule. Our study reveled that the second sphere residue His228 is responsible for the water deprotonation through hydrogen bonding. The spectroscopic and crystallographic data showed the H228Y mutation binds ferric iron instead of native zinc metal and the active site water is replaced by the Tyr228 residue ligation. Thus, we concluded that, H228Y not only plays a role of stabilizing and deprotonating the active site water but also is an essential residue on metal selectivity.