Portland State University. Department of Chemistry
Term of Graduation
Date of Publication
Doctor of Philosophy (Ph.D.) in Chemistry
Adenosylmethionine, Transfer RNA
1 online resource (2, xiv, 196 pages)
Queuosine is a hypermodified nucleoside located in the wobble position of bacterial and eukaryotic tRNAs coding for Asp, Tyr, His and Asn. The biosynthesis involves the participation of S-adenosyl-methionine:tRNA ribosyltransferase-isomerase (QueA) and a GTP Cyclohydrolase-I. QueA catalyzes the transfer and isomerization of the ribosyl moiety from AdoMet to preQ1 modified tRNA. Substrate analogs of AdoMet were used to elucidate important substrate-enzyme interactions and to test key steps in the proposed chemical mechanism. Replacing AdoMet with SeAdoMet had little effect upon substrate binding but exhibited 30-fold reduction in kcat, consistent with deprotonation at C-5' as the first catalytic step. 7-deazaAdoMet failed to function as a substrate of QueA, but exhibited a Ki that was only slightly higher than the K m for AdoMet, suggesting that N-7 is critical for catalysis but not substrate binding. Neither the 2'- or 3'-deoxyAdoMet exhibited activity with QueA, however both analogs had a Ki of only 2-fold higher than the Km of AdoMet.
Reported here is the identification and characterization of the COG1469 protein family as a novel Type-I GTP cyclohydrolase (GCYH) that catalyzes the conversion of GTP to 7,8-dihydroneopterin-triphosphate in ∼20% of bacteria and most archaea. The COG1469 proteins and the genes that encode them were renamed GCYH-IB and folE-2, respectively, whereas the canonical cyclohydrolase, was renamed GCYH-IA. B. subtilis and N. gonorrhoeae GCYH-IB are homotrimers that were maximally active in the presence of manganese. GDP also functioned as a substrate; however the removal of the γ-phosphate resulted in a ∼30-fold decrease in kcat/Km. Inhibition analysis with N. gonorrhoeae GCYH-IB demonstrated that 8-oxoGTP functioned as a potent inhibitor with a K145-fold lower than the Km for GTP. Although the 7-deazaGTP did not function as a substrate for GCYH-IB, it exhibited a Ki 7-fold higher than the Km for GTP. The Ki for 2'-deoxyGTP was 24-fold higher than the Km of GTP. H245 of N. gonorrhoeae GCYH-IB was believed to function analogous to that of H179 of E. coli FolE which facilitates the elimination of C-8 from GTP. However, H245 mutants were still able to catalyze the elimination of C-8, suggesting that H245 is not involved in the deformylation of GTP.
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Bonnett, Shilah Amal, "The Characterization of tRNA Modifying Enzymes S-adenosylmethionine : tRNA Ribosyltransferase-isomerase (QueA) and a novel Type I GTP cyclohydrolase" (2007). Dissertations and Theses. Paper 6180.