Bacterial tRNA adenosine deaminases (TadAs) catalyze the hydrolytic deamination of adenosine to inosine at the wobble position of tRNA(Arg2), a process that enables this single tRNA to recognize three different arginine codons in mRNA. In addition, inosine is also introduced at the wobble position of multiple eukaryotic tRNAs. The genes encoding these deaminases are essential in bacteria and yeast, demonstrating the importance of their biological activity. Here we report the crystallization and structure determination to 2.0 A of Staphylococcus aureus TadA bound to the anticodon stem-loop of tRNA(Arg2) bearing nebularine, a non-hydrolyzable adenosine analog, at the wobble position. The cocrystal structure reveals the basis for both sequence and structure specificity in the interactions of TadA with RNA, and it additionally provides insight into the active site architecture that promotes efficient hydrolytic deamination.

No abstract

The structure of GtfB places it in a growing group of glycosyltransferases, including Escherichia coli MurG and a beta-glucosyltransferase from T4 phage, which together form a subclass of the glycosyltransferase superfamily and give insights into the recognition of the NDP-sugar and aglycone cosubstrates. A single major interdomain linker between the N- and C- terminal domains suggests that reprogramming of sugar transfer or aglycone recognition in the antibiotic glycosyltransferases, including the glycopeptide and also the macrolide antibiotics, will be facilitated by this structural information.

During the biosynthesis of the vancomycin-class antibiotic chloroeremomycin, TDP-epi-vancosaminyltransferase GtfA catalyzes the attachment of 4-epi-vancosamine from a TDP donor to the ␤-OHTyr-6 of the aglycone cosubstrate. Glycosyltransferases from this pathway are potential tools for the combinatorial design of new antibiotics that are effective against vancomycin-resistant bacterial strains. These enzymes are members of the GT-B glycosyltransferase superfamily, which share a homologous bidomain topology. We present the 2.8-Å crystal structures of GtfA complexes with vancomycin and the natural monoglycosylated peptide substrate, representing the first direct observation of acceptor substrate binding among closely related glycosyltransferases. The acceptor substrates bind to the N-terminal domain such that the aglycone substrate's reactive hydroxyl group hydrogen bonds to the side chains of Ser-10 and Asp-13, thus identifying these as residues of potential catalytic importance. As well as an open form of the enzyme, the crystal structures have revealed a closed form in which a TDP ligand is bound at a donor substrate site in the interdomain cleft, thereby illustrating not only binding interactions, but the conformational changes in the enzyme that accompany substrate binding.T he glycopeptide antibiotics of the vancomycin family are clinically important for the treatment of Gram-positive bacterial infections. These natural products are derived from an oxidatively crosslinked heptapeptide scaffold, which is differentially glycosylated (Fig. 1). The number, identity, and position of these sugar moieties influence bioactivity, both by increasing solubility and by mediating important binding interactions. Glycosylation of the aglycone core is performed in the final stages of biosynthesis by a series of structurally homologous, dedicated glycosyltransferase (Gtf) tailoring enzymes, using an NDP-hexose substrate as a sugar donor (1).The emergence of vancomycin resistance has created an urgent need for novel antibiotics active against resistant bacterial strains. Modification of the carbohydrate moieties on vancomycin has been shown to overcome resistance (2, 3), possibly through different modes of action (3). Because of the complex chemistry involved, vancomycin analogs are prohibitively difficult to produce synthetically. However, the Gtfs from the natural biosynthetic pathways are promising tools for the combinatorial design of new antibiotics with enhanced potency or novel activity by reprogramming them to accept alternative aglycone scaffolds or NDP-sugar donors. Previous studies have demonstrated that some Gtfs from the vancomycin and chloroeremomycin pathways can effectively use nonnatural substrates to generate new compounds (1, 4, 5). Moreover, if the structural determinants of substrate specificity can be understood, the directed genetic modification of these enzymes could be exploited to yield a broader diversity of products.We previously reported the x-ray crystal structure of the first of these enzymes, Gt...