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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2009-12-7250 |
| Date | 2009 December 1900 |
| Creators | Hall, Richard Stuart |
| Contributors | Raushel, Frank M. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | application/pdf |
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