Formycin A is a potent purine nucleoside antibiotic with a C-glycosidic linkage between the ribosyl moiety and the pyrazolopyrimidine base. Herein, a cosmid is identified from the Streptomyces kaniharaensis genome library that contains the for gene cluster responsible for the biosynthesis of formycin. Subsequent gene deletion experiments and in vitro characterization of the forBCH gene products established their catalytic functions in formycin biosynthesis. Results also demonstrated that PurH from de novo purine biosynthesis plays a key role in pyrazolopyrimidine formation during biosynthesis of formycin A. The participation of PurH in both pathways represents a good example of how primary and secondary metabolism are interlinked.
It has been hypothesized that mitochondria evolved from a bacterial ancestor that initially became established in a protoeukaryotic cell as an endosymbiont. Here we model this first stage of mitochondrial evolution by engineering endosymbiosis between E. coli and the budding yeast S. cerevisiae. Fusion of yeast with E. coli ectopically expressing several genes from unrelated, intracellular bacteria was key for establishing endosymbiosis. ADP/ATP translocase‐expressing E. coli provided an energy source for a respiration‐deficient cox2 yeast mutant, enabling growth of yeast‐E. coli chimera on a non‐fermentable carbon source. Similarly, yeast provided a source of thiamin or NAD to an E. coli thiamin or NAD auxotroph respectively. Expression of several SNARE‐like protein on the surface of E. coli was also required to prevent lysosomal degradation. The engineered yeast‐E. coli chimeras sustained growth on selection medium in containing the antibiotic carbenicillin indicating the presence of intracellular E. coli which supports the growth by ATP synthesis on selection medium. Further, sf‐gfp expressing E. coli endosymbionts could be observed in the yeast by super resolution fluorescence microscopy after more than 40 doublings. This readily manipulated system should allow us to experimentally delineate host‐endosymbiont adaptations that occurred during evolution of the current much reduced mitochondrial genome This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.
C-Nucleosides are characterized by aC À Cr ather than aC À Nl inkage between the heterocyclic base and the ribofuranose ring. While the biosynthesis of pseudouridine-C
DesII is a radical SAM enzyme that catalyzes the C4-deamination of TDP-4-amino-4,6-dideoxyglucose via a C3 radical intermediate. However, if the C4 amino group is replaced with a hydroxyl (TDP-quinovose), the hydroxyl at C3 is oxidized to a ketone with no C4-dehydration. It is hypothesized that hyperconjugation between the C4 C-N/O bond and the partially filled porbital at C3 of the radical intermediate modulates the degree to which elimination competes with dehydrogenation. To investigate this hypothesis, the reaction of DesII with the C4-epimer of TDPquinovose (TDP-fucose) was examined. The majority of the reaction results in the formation of TDP-6-deoxygulose and likely regeneration of TDP-fucose. The remainder of the substrate radical partitions roughly equally between C3-dehydrogenation and C4-dehydration. Thus, changing the stereochemistry at C4 permits a more balanced competition between elimination and dehydrogenation. KeywordsEnzyme catalysis; Radical SAM; Alcohols; Biosynthesis The radical S-adenosylmethionine (SAM) enzyme DesII from Streptomyces venezuelae catalyzes the redox-neutral deamination of TDP-4-amino-4,6-dideoxy-D-glucose (1) to generate TDP-4,6-dideoxy-3-keto-D-glucose (2, see Scheme 1). 1,2 In its biological context, the deamination of 1 is the key reaction in the biosynthesis of TDP-desosamine (3), which is an essential component of many macrolide antibiotics. [2][3][4] This deamination reaction is radical-mediated and is initiated via hydrogen atom abstraction from the substrate by a 5′-deoxyadenosyl radical. The latter is derived from reductive homolysis of SAM by an active site [4Fe-4S] 1+ cluster and represents the hallmark of radical SAM enzymology. 5 Two general mechanisms have been proposed for the DesII-catalyzed deamination as shown in Figure 1. 1,4 In both cases, the p-orbital harboring the unpaired electron at C3 of the radical intermediate 6 must overlap productively with the C-N σ-system at C4 in order to ** This work was supported by grants from the National Institutes of Health (GM035906) and the Welch Foundation (F-1511). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript facilitate either 1,2-migration (6→7→9/10→2) or direct elimination (6→8→9→2) of the adjacent amino group. In this regard, DesII is highly reminiscent of ethanolamine ammonia lyase (EAL), which catalyzes the deamination of ethanolamine albeit using a 5′-deoxyadenosyl radical produced from the homolysis of adenosylcobalamin rather than SAM. 6 Although the chemistry of DesII is also very similar to the dehydration of 1,2-diols by the B 12 -dependent dioldehydratases, 7 no elimination of the C2 hydroxyl from 1 is observed during the catalytic cycle of DesII.DesII can also accept TDP-D-quinovose (4) as a substrate where the C4 amino group of 1 is replaced with a hydroxyl group. However, DesII does not catalyze elimination of the C4 hydroxyl from 4 to produce 2, but rather oxidation of the C3 hydroxyl group to yield 5 (see Scheme 1). 1 This second, dehydrogenase activity of D...
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