Yeasts, which have been a component of the human diet for at least 7000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for Bacteroides thetaiotaomicron (Bt), a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by Bt presents a ‘selfish’ model for the catabolism of this recalcitrant polysaccharide. This report shows how a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet.

The syntheses of ATP, GTP, UTP, and CTP with deuterium labels on the 3‘, 4‘, and 5‘ carbons (2−5) is described. A combination of chemical and enzymatic synthesis is used where d,l-ribose-3,4,5,5‘-d 4 (±1) is first produced from glycerol-d 8 by chemical methods, and then the four 3‘,4‘,5‘,5‘-labeled NTPs (2−5) are prepared from (− 1) using enzymes from the purine salvage and pyrimidine biosynthetic metabolic pathways. New procedures were developed for the large scale preparation of GTP and CTP, and existing procedures were modified for the preparation of ATP and UTP. A 30-nucleotide RNA derived from the HIV-2 TAR RNA was prepared with unlabeled NTPs and deuterated NTPs (2−5) to illustrate the dramatic effects of deuteration on the NMR spectra of RNA. The NOESY spectra of the deuterated RNA exhibits greatly reduced spectral crowding compared to that of the unlabeled RNA, and assignment of NOEs to the H2‘ protons is simplified due to the specific deuteration pattern. Also, the nonselective T 1 and T 2 relaxation rates were measured for the deuterated RNA and found to be approximately twice as long as the T 1 and T 2 relaxation rates of the unlabeled RNA. The spectral simplification and improved relaxation properties of the deuterated RNA should prove useful in the study of large RNAs by NMR.

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The highly selective tobacco etch virus Nla protease (TEV protease) can be used to remove affinity tags with mutated cleavage sites to produce proteins for site‐specific labeling and chemoselective ligations (see scheme). This provides a simple, efficient method for producing proteins with N‐terminal cysteines for proteomic applications.

The enzymatic conversion of glucose into ATP, GTP, UTP, and CTP with several different isotopic labeling patterns is described. Enzymes of the pentose phosphate pathway and enzyme-catalyzed hydrogen exchange were used to convert three types of isotopically labeled glucose into [1‘,2‘,3‘,4‘,5‘,5‘-2H6]NTPs (1−4), [3‘,4‘,5‘,5‘-2H4]UTP (5), [1‘,2‘,3‘,4‘,5‘-13C5]NTPs (6−9), and [3‘,4‘,5‘,5‘-2H4-1‘,2‘,3‘,4‘,5‘-13C5]NTPs (10−13), which were then used to synthesize a 30 nucleotide HIV TAR RNA. Representative NOESY and HSQC spectra were acquired to demonstrate the utility of the new labeling patterns. The spectral editing afforded by 2H and 13C labeling dramatically simplifies the crowded NOESY and HSQC spectra of RNA molecules. The synthetic methods described here will permit the preparation of several specifically deuterated and/or 13C-labeled forms of RNA which should be useful in NMR structural studies of large RNAs.