First Advisor

Dirk Iwata-Reuyl

Term of Graduation

Spring 2007

Date of Publication


Document Type


Degree Name

Doctor of Philosophy (Ph.D.) in Environmental Sciences and Resources: Chemistry


Environmental Sciences and Resources




Biosynthesis, Enzymology, Nucleosides, RNA editing



Physical Description

1 online resource (2, xii, 213 pages)


Queuosine (Q) is a modified nucleoside found at the wobble position of bacterial and eukaryotic transfer RNAs (tRNAs) that are specific for the amino acids tyrosine, histidine, aspartate and asparagine. A recently discovered enzyme in the biosynthetic pathway of Q, the NADPH-dependent 7-cyano-7-deazaguanine oxidoreductase (QueF), carries out the two-fold, four-electron reduction of Q precursor preQ0 to preQ1 and represents the first example of the enzymatic conversion of the nitrile functional group to an amine. Presented herein are kinetic, spectroscopic, mutational, biophysical, and isotope labeling studies directed at the elucidation of the chemical and kinetic mechanisms of this new class of protein.

Steady-state kinetic analysis using a NADPH-linked continuous assay provided the kinetic parameters Km(NADPH) = 19 ± 2 μM and kcat = 0.69 ± 0.02 min-1. To determine the kinetic parameters of preQ0, a fluorescence assay that was used to follow the formation of NADP+ as an alkaline degradation product was optimized, and this method gave the kinetic constants Km (preQ0) = 0.237 ± 0.045 μM, and k cat = 0.66 ± 0.04 min-1.

Titrations of enzyme with preQ0, inactivation and protection studies with iodoacetamide, and site-directed mutagenesis of a conserved cysteine residue (Cys55 in Bacillus subtilis), followed by the biochemical and biophysical analysis of the resulting protein products suggest covalent catalysis is employed by QueF, with Cys55 serving as the catalytic nucleophile to form a covalent thioimide adduct.

The mechanism of hydride transfer from NADPH to preQ0 was addressed using isotope labeling studies. The data obtained from 1 H-NMR, ESI-MS and steady-state kinetic analysis of QueF suggests that the protein promotes the stereospecific transfer of the pro-R hydride of NADPH.

Mutational and kinetic studies are also described for the substitution of a conserved glutamate (Glu97 in B. subtilis). As evidenced by 25-fold to 280-fold increases in Km (preQ 0) values, the results show this residue is critical for preQ0 recognition and binding. In addition, the kinetics revealed 13-fold to 20-fold decreases in kcat, suggesting substitution of Glu97 also impacts chemistry at the active site.

These data provide insights into the active-site chemistry of the QueF mediated nitrile reduction and a chemical mechanism consistent with our results is proposed.


In Copyright. URI:

This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).


If you are the rightful copyright holder of this dissertation or thesis and wish to have it removed from the Open Access Collection, please submit a request to and include clear identification of the work, preferably with URL.

Persistent Identifier