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Results: 3

1.
Fig. 1.

Fig. 1. From: Discovery of epoxyqueuosine (oQ) reductase reveals parallels between halorespiration and tRNA modification.

Biosynthetic pathway for production of queuosine with the oQ reductase step elucidated in this manuscript boxed. The block arrow denotes a series of steps analogous to purine salvage/repair, which convert the 7-cyano-7-deazaguanine base to toyocamycin.

Zachary D. Miles, et al. Proc Natl Acad Sci U S A. 2011 May 3;108(18):7368-7372.
2.
Fig. 2.

Fig. 2. From: Discovery of epoxyqueuosine (oQ) reductase reveals parallels between halorespiration and tRNA modification.

Analysis of RNA from Keio collection deletion strains for presence and absence of Q and oQ. A shows a representative UV-visible trace of the RNA samples, highlighting the region where oQ and Q elute. Wild-type E. coli contain very little oQ when grown in LB. B shows extracted ion chromatograms of wild-type and deletion strains corresponding to each step in the pathway. Deletion of any gene up to and including Tgt leads to complete absence of oQ (m/z = 426) or Q (m/z = 410). Deletion of queA leads to accumulation of preQ1 nucleoside (m/z = 312), as would be expected (see Fig. 1). Deletion of queG leads to complete absence of Q and accumulation of oQ. A seven-point data smoothing algorithm was applied to all of the extracted ion chromatograms.

Zachary D. Miles, et al. Proc Natl Acad Sci U S A. 2011 May 3;108(18):7368-7372.
3.
Fig. 3.

Fig. 3. From: Discovery of epoxyqueuosine (oQ) reductase reveals parallels between halorespiration and tRNA modification.

Activity assays with oQ reductase. Incubation of oQ reductase in the presence of methyl viologen, dithionite, and B12 leads to time-dependent disappearance of the oQ and appearance of Q (A). In the time course, the areas of the extracted ion chromatograms at m/z = 426 or 410, corresponding to oQ or Q, respectively, were measured and plotted. The intensity of the peaks at each time point were referenced to the intensities of the unreacted substrate (100% oQ) or full conversion (100% Q). B12 stimulates activity by approximately 30%. Control experiments show that methyl viologen and dithionite are absolutely required as shown in extracted ion chromatograms in B. A seven-point data smoothing algorithm was applied to all of the extracted ion chromatograms.

Zachary D. Miles, et al. Proc Natl Acad Sci U S A. 2011 May 3;108(18):7368-7372.

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