Background: Schwanniomyces occidentalis ␤-fructofuranosidase synthesizes 6-kestose and 1-kestose, prebiotics that stimulate beneficial bacteria. Results:The ␤-sandwich domain is involved in substrate binding, and the Gln-228/Asn-254 pair plays a crucial role in acceptor/ donor substrate binding and product specificity. Conclusion: First evidence of ␤-sandwich domain functionality. Variants with new specificities, synthesizing neokestose, were obtained. Significance: Understanding the molecular mechanism that regulates product specificity is crucial for designing improved new enzymes.

Background: Invertase is a fundamental enzyme for sugar metabolism in yeast and a classical model in early biochemical studies. Results: Invertase shows an unusual octameric quaternary structure composed of two types of dimers. Conclusion: A peculiar pattern of monomer assembly through non-catalytic domain interactions determines invertase specificity. Significance: Unraveling the structural features that rule enzyme modularity casts new light on protein-carbohydrate recognition.

The Type VI secretion system (T6SS) is a bacterial nanomachine which delivers toxic effectors to kill competitors or subvert some of their key functions. Here we use tra nsposon d irected i nsertion-site s equencing (TraDIS) to identify T6SS toxins associated with the H1-T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa . This approach identified several putative toxin-immunity pairs, including Tse8-Tsi8. Full characterization of this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis in bacteria that lack either one, or both, of the asparagine and glutamine tRNA synthases. Biochemical characterization of the interactions between Tse8 and the transamidosome components GatA, GatB and GatC suggests that the presence of Tse8 alters the fine-tuned stoichiometry of the transamidosome complex, and in vivo assays demonstrate that Tse8 limits the ability of prey cells to synthesize proteins. These data expand the range of cellular components targeted by the T6SS by identifying a T6SS toxin affecting protein synthesis proteins and validate the use of a TraDIS-based global genomics approach to expand the repertoire of T6SS toxins in T6SS-encoding bacteria.

Glycosyltransferases (GTs) are a key family of enzymes that catalyze the synthesis of glycosidic bonds in all living organisms. The reaction involves the transfer of a glycosyl moiety and can proceed with retention or inversion of the anomeric configuration. To date, the catalytic mechanism of retaining GTs is a topic of great controversy, particularly for those enzymes containing a putative nucleophilic residue in the active site, for which the occurrence of a double-displacement mechanism has been suggested. We report native ternary complexes of the retaining glycosyltransferase α-1,3-galactosyltransferase (α3GalT) from Bos taurus, which contains such a nucleophile in the active site, in a productive mode for catalysis in the presence of its sugar donor UDP-Gal, the acceptor substrate lactose, and the divalent cation cofactor. This new experimental evidence supports the occurrence of a front-side substrate-assisted S i-type reaction for α3GalT, and suggests a conserved common catalytic mechanism among retaining GTs.