Improved sequencing technologies have created an explosion of sequence information that is analyzed and proteins are annotated automatically. Annotations are made based on similarity scores to previously annotated sequences, so one misannotation is propagated throughout databases and the number of misannotated proteins grows with the number of sequenced genomes. A systematic approach to correctly identify the function of proteins in the amidohydrolase superfamily is described in this work using Clusters of Orthologous Groups of proteins as defined by NCBI. The focus of this work is COG1816, which contains proteins annotated, often incorrectly, as adenosine deaminase enzymes. Sequence similarity networks were used to evaluate the relationship between proteins. Proteins previously annotated as adenosine deaminases: Pa0148 (Pseudomonas aeruginosa PAO1), AAur_1117 (Arthrobacter aurescens TC1), Sgx9403e and Sgx9403g, were purified and their substrate profiles revealed that adenine and not adenosine was a substrate for these enzymes. All of these proteins will deaminate adenine with values of kcat/Km that exceed 105 M-1s-1. A small group of enzymes similar to Pa0148 was discovered to catalyze the hydrolysis of N-6-substituted adenine derivatives, several of which are cytokinins, a common type of plant hormone. Patl2390, from Pseudoalteromonas atlantica T6c, was shown to hydrolytically deaminate N-6-isopentenyladenine to hypoxanthine and isopentenylamine with a kcat/Km of 1.2 x 107 M^-1 s^-1. This enzyme does not catalyze the deamination of adenine or adenosine. Two small groups of proteins from COG1816 were found to have 6-aminodeoxyfutalosine as their true substrate. This function is shared with 2 small groups of proteins closely related to guanine and cytosine deaminase from COG0402. The deamination of 6-aminofutalosine is part of the alternative menaquinone biosynthetic pathway that involves the formation of futalosine. 6-Aminofutalosine is deaminated with a catalytic effeciency of 105 M-1s-1 or greater, Km’s of 0.9 to 6.0 µM and kcat’s of 1.2 to 8.6 s-1. Another group of proteins was shown to deaminate cyclic- 3’, 5’ -adenosine monophosphate (cAMP) to produce cyclic-3’, 5’-inosine monophosphate, but will not deaminate adenosine, adenine or adenosine monophosphate. This protein was cloned from a human pathogen, Leptospira interrogans. Deamination may function in regulating the signaling activities of cAMP.

The amidohydrolase superfamily is a functionally diverse group of evolutionarily related proteins which utilize metal cofactors in the activation of a hydrolytic water molecule and in the stabilization of the resulting tetrahedral intermediate. Members of this superfamily have been described which use one or two divalent transition metals. These metal cofactors are located in either or both of two active-site metal binding centers which are labeled as the Ma and MB sites. The goal of this research was to elucidate the nature of the reactions catalyzed by Ma and MB mononuclear members of the amidohydrolase superfamily. This was approached through comprehensive mechanistic evaluations of two enzymes which utilized the different metal sites. Nacetyl- D-glucosamine-6-phosphate deacetylase from E. coli (NagA) and cytosine deaminase from E. coli (CDA) served as models for mononuclear amidohydrolase superfamily enzymes which have evolved to utilize a single B-metal and a single a-metal for hydrolysis, respectively. This research elucidated the different properties imparted by the distinct a and B active sites and the specific interactions utilized by the enzymes for substrate binding and catalysis. These studies led to the eventual proposal of detailed chemical mechanisms and the identification of rate determining steps. Knowledge of sequence-function relationships was applied toward the discovery of function for enzymes related to cytosine deaminase and guanine deaminase. The first group of enzymes investigated was proposed to catalyze the fourth step in riboflavin and coenzyme F420 biosynthesis in Achaea. Three putative deaminases; Mm0823 from Methanosarcina mazei, MmarC7_0625 from Methanococcus maripaludis C7 and Sso0398 from Sulfolobus solfataricus were cloned and expressed. These proteins proved to be intractably insoluble. A second set of enzymes, Pa0142 from Pseudomonas aeruginosa PA01 and SGX-9236e (with crystal structure PDB: 3HPA) were found to catalyze the novel deamination of 8-oxoguanine, a mutagenic product of DNA oxidation. 9236e was cloned from an unidentified environmental sample of the Sargasso Sea. The closest homolog (98% identical) is Bcep18194_A5267 from Burkholderia sp. 383. Additionally, it was discovered that the proteins SGX-9339a (with crystal structure PDB: 2PAJ) and SGX-9236b catalyzed the deamination of isoxanthopterin and pterin-6- carboxylate in a poorly characterized folate degradation pathway. These enzymes were also from unknown environmental samples of the Sargasso Sea. The closest homolog of 9339a (88% identical) is Bxe_A2016 from Burkholderia xenovorans LB400. The closest homolog of 9236b (95% identical) is Bphyt_7136 from Burkholderia phytofirmans PsJN.