Dataset Information

Fidelity and Promiscuity of a Mycobacterial Glycosyltransferase.

ABSTRACT: Members of the genus Mycobacterium cause devastating human diseases, including tuberculosis. Mycobacterium tuberculosis can resist some antibiotics because of its durable and impermeable cell envelope. This barrier is assembled from saccharide building blocks not found in mammals, including galactofuranose (Galf). Within the cell envelope, Galf residues are linked together to afford an essential polysaccharide, termed the galactan. The formation of this polymer is catalyzed by the glycosyltransferase GlfT2, a processive carbohydrate polymerase, which generates a sequence-specific polysaccharide with alternating regioisomeric ?(1-5) and ?(1-6) Galf linkages. GlfT2 exhibits high fidelity in linkage formation, as it will terminate polymerization rather than deviate from its linkage pattern. These findings suggest that GlfT2 would prefer an acceptor with a canonical alternating ?(1-5) and ?(1-6) Galf sequence. To test this hypothesis, we devised a synthetic route to assemble oligosaccharides with natural and non-natural sequences. GlfT2 could elongate each of these acceptors, even those with non-natural linkage patterns. These data indicate that the glycosyltransferase is surprisingly promiscuous in its substrate preferences. However, GlfT2 did favor some substrates: it preferentially acted on those in which the lipid-bearing Galf residue was connected to the sequence by a ?(1-6) glycosidic linkage. The finding that the relative positioning of the lipid and the non-reducing end of the acceptor influences substrate selectivity is consistent with a role for the lipid in acceptor binding. The data also suggest that the fidelity of GlfT2 for generating an alternating ?(1-5) and ?(1-6) pattern of Galf residues arises not from preferential substrate binding but during processive elongation. These observations suggest that inhibiting the action of GlfT2 will afford changes in cell wall structure.

SUBMITTER: Yamatsugu K

PROVIDER: S-EPMC5349768 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Fidelity and Promiscuity of a Mycobacterial Glycosyltransferase.

Yamatsugu Kenzo K Splain Rebecca A RA Kiessling Laura L LL

Journal of the American Chemical Society 20160713 29

Members of the genus Mycobacterium cause devastating human diseases, including tuberculosis. Mycobacterium tuberculosis can resist some antibiotics because of its durable and impermeable cell envelope. This barrier is assembled from saccharide building blocks not found in mammals, including galactofuranose (Galf). Within the cell envelope, Galf residues are linked together to afford an essential polysaccharide, termed the galactan. The formation of this polymer is catalyzed by the glycosyltransf ...[more]

PMID: 27302377

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Project description:Most bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNAGln and tRNAAsn. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance. Furthermore, we identified clinical isolates of the globally important pathogen Mycobacterium tuberculosis with partial loss-of-function mutations in gatA, and demonstrated that these mutations result in high, specific rates of translational error and increased rifampin tolerance. However, the mechanisms by which these clinically derived mutations in gatA impact GatCAB function were unknown. Here, we describe biochemical and biophysical characterization of M. tuberculosis GatCAB, containing either wild-type gatA or one of two gatA mutants from clinical strains. We show that these mutations have minimal impact on enzymatic activity of GatCAB; however, they result in destabilization of the GatCAB complex as well as that of the ternary asparaginyl-transamidosome. Stabilizing complex formation with the solute trehalose increases specific translational fidelity of not only the mutant strains but also of wild-type mycobacteria. Therefore, our data suggest that alteration of GatCAB stability may be a mechanism for modulation of translational fidelity. IMPORTANCE Most bacteria use a two-step indirect pathway to aminoacylate tRNAGln and tRNAAsn, despite the fact that the indirect pathway consumes more energy and is error prone. We have previously shown that the higher protein synthesis errors from this indirect pathway in mycobacteria allow adaptation to hostile environments such as antibiotic treatment through generation of novel alternate proteins not coded by the genome. However, the precise mechanisms of how translational fidelity is tuned were not known. Here, we biochemically and biophysically characterize the critical enzyme of the Mycobacterium tuberculosis indirect pathway, GatCAB, as well as two mutant enzymes previously identified from clinical isolates that were associated with increased mistranslation. We show that the mutants dysregulate the pathway via destabilizing the enzyme complex. Importantly, increasing stability improves translational fidelity in both wild-type and mutant bacteria, demonstrating a mechanism by which mycobacteria may tune mistranslation rates.

Dataset Information

Fidelity and Promiscuity of a Mycobacterial Glycosyltransferase.

Publications

Fidelity and Promiscuity of a Mycobacterial Glycosyltransferase.

Similar Datasets

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