High throughput sequencing technology and availability of this information has changed the way enzyme families can be studied. Sequence information from large public databases such as GenBank and UniProtKB can easily retrieved for the purpose of identifying unique enzymatic activities. The strategy adopted for this study is to identify characterized enzymes and the sequence features which give rise to their substrate specificity. Homologues of these enzymes are retrieved, and any active site variations can be readily identified. Cluster of Orthologous Groups (cog) 0402 is a family of enzymes which comprise a portion of the amidohydrolase superfamily. This group catalyzes a deamination reaction, releasing free ammonia and replacing it with a tautomerized oxygen. Cog0402 is most well known for guanine and cytosine deaminase, however other functions exist. One such function was that of S-adenosylhomocysteine deaminase, which was related to a large group of uncharacterized enzymes. These enzymes were predicted by us to deaminate 5'-modified adenosines. The enzymes were physically characterized these predictions were confirmed and a 5'-deoxyadenosine deaminase was discovered in addition to an 8-oxoadenine deaminase. During this study it was noted that background isoguanine deaminase activity was found at appreciable rates in E. coli. This activity was purified and identified using nanoLC-MS/MS and found to be caused by E. coli cytosine deaminase. E. coli cytosine deaminase itself is found in a cluster of uncharacterized enzymes with a single amino acid difference in the active site. Representative enzymes were purified and a 5-methylcytosine deaminase was discovered. This enzyme is capable of rescuing thymine auxotrophs in the presence of 5-methylcytosine, and will confer sensitivity to 5-fluorocytosine. Finally, an enzyme distantly related to cytosine deaminase was purified and found to be a unique pterin deaminase. It was most efficient for oxidized pterin rings and would accept a variety of substituents on the C6 positions. Futhermore, it was thought to catalyze the first step of an undescribed pterin degradation pathway.
